Ablation of sarcolipin enhances sarcoplasmic reticulum calcium transport and atrial contractility

Sarcolipin is a novel regulator of cardiac sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) and is expressed abundantly in atria. In this study we investigated the physiological significance of sarcolipin in the heart by generating a mouse model deficient for sarcolipin. The sarcolipin-null mice do not show any developmental abnormalities or any cardiac pathology. The absence of sarcolipin does not modify the expression level of other Ca2+ handling proteins, in particular phospholamban, and its phosphorylation status. Calcium uptake studies revealed that, in the atria, ablation of sarcolipin resulted in an increase in the affinity of the SERCA pump for Ca2+ and the maximum velocity of Ca2+ uptake rates. An important finding is that ablation of sarcolipin resulted in an increase in atrial Ca2+ transient amplitudes, and this resulted in enhanced atrial contractility. Furthermore, atria from sarcolipin-null mice showed a blunted response to isoproterenol stimulation, implicating sarcolipin as a mediator of beta-adrenergic responses in atria. Our study documented that sarcolipin is a key regulator of SERCA2a in atria. Importantly, our data demonstrate the existence of distinct modulators for the SERCA pump in the atria and ventricles.     

Involvement of the sarcoplasmic reticulum calcium pump in myocardial contractile dysfunction: Comparison between chronic pressure-overload and stunning

Summary Acute as well as chronic forms of heart failure involve mechanical dysfunction during systole and/or diastole. The rapid Ca²⁺ release from and Ca²⁺ reuptake into the tubuli of the sarcoplasmic reticulum are processes that critically determine normal systolic and diastolic myocardial function, which explains why in the last fifteen years so much attention has been paid to understand the performance of the sarcoplasmic reticulum Ca²⁺ pump during myocardial contractile dysfunction. In this communication we have reviewed the literature data on sarcoplasmic reticulum Ca²⁺ pump function in the chronically pressure-overloaded hypertrophied and stunned (post-ischemic reversibly injured) myocardium in the light of some new data from our laboratory. Results on the pressure-overloaded hypertrophied myocardium provide evidence that impaired relaxation is most likely due to a low capacity of the sarcoplasmic reticulum to pump Ca²⁺, a consequence of a lower density of Ca²⁺-pumping sites within the sarcotubular membranes. Contractile dysfunction in stunned myocardium is accompanied by an upregulation of the sarcoplasmic reticulum Ca²⁺ ATPase gene resulting in a slight increase of the Ca²⁺ pumping activity. The latter increase is likely an adaptive response of the reversibly injured myocardium which may contribute to the slow recovery of contractile function.     

CaMKII inhibition targeted to the sarcoplasmic reticulum inhibits frequency-dependent acceleration of relaxation and Ca2+ current facilitation

Cardiac Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in heart has been implicated in Ca(2+) current (I(Ca)) facilitation, enhanced sarcoplasmic reticulum (SR) Ca(2+) release and frequency-dependent acceleration of relaxation (FDAR) via enhanced SR Ca(2+) uptake. However, questions remain about how CaMKII may work in these three processes. Here we tested the role of CaMKII in these processes using transgenic mice (SR-AIP) that express four concatenated repeats of the CaMKII inhibitory peptide AIP selectively in the SR membrane. Wild type mice (WT) and mice expressing AIP exclusively in the nucleus (NLS-AIP) served as controls. Increasing stimulation frequency produced typical FDAR in WT and NLS-AIP, but FDAR was markedly inhibited in SR-AIP. Quantitative analysis of cytosolic Ca(2+) removal during [Ca(2+)](i) decline revealed that FDAR is due to an increased apparent V(max) of SERCA. CaMKII-dependent RyR phosphorylation at Ser2815 and SR Ca(2+) leak was both decreased in SR-AIP vs. WT. This decrease in SR Ca(2+) leak may partly balance the reduced SERCA activity leading to relatively unaltered SR-Ca(2+) load in SR-AIP vs. WT myocytes. Surprisingly, CaMKII regulation of the L-type Ca(2+) channel (I(Ca) facilitation and recovery from inactivation) was abolished by the SR-targeted CaMKII inhibition in SR-AIP mice. Inhibition of CaMKII effects on I(Ca) and RyR function by the SR-localized AIP places physical constraints on the localization of these proteins at the junctional microdomain. Thus SR-targeted CaMKII inhibition can directly inhibit the activation of SR Ca(2+) uptake, SR Ca(2+) release and I(Ca) by CaMKII, effects which have all been implicated in triggered arrhythmias.     

Ca2+/calmodulin kinase II increases ryanodine binding and Ca2+-induced sarcoplasmic reticulum Ca2+ release kinetics during beta-adrenergic stimulation

We aimed to define the relative contribution of both PKA and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) cascades to the phosphorylation of RyR2 and the activity of the channel during beta-adrenergic receptor (betaAR) stimulation. Rat hearts were perfused with increasing concentrations of the beta-agonist isoproterenol in the absence and the presence of CaMKII inhibition. CaMKII was inhibited either by preventing the Ca(2+) influx to the cell by low [Ca](o) plus nifedipine or by the specific inhibitor KN-93. We immunodetected RyR2 phosphorylated at Ser2809 (PKA and putative CaMKII site) and at Ser2815 (CaMKII site) and measured [(3)H]-ryanodine binding and fast Ca(2+) release kinetics in sarcoplasmic reticulum (SR) vesicles. SR vesicles were isolated in conditions that preserved the phosphorylation levels achieved in the intact heart and were actively and equally loaded with Ca(2+). Our results demonstrated that Ser2809 and Ser2815 of RyR2 were dose-dependently phosphorylated under betaAR stimulation by PKA and CaMKII, respectively. The isoproterenol-induced increase in the phosphorylation of Ser2815 site was prevented by the PKA inhibitor H-89 and mimicked by forskolin. CaMKII-dependent phosphorylation of RyR2 (but not PKA-dependent phosphorylation) was responsible for the beta-induced increase in the channel activity as indicated by the enhancement of the [(3)H]-ryanodine binding and the velocity of fast SR Ca(2+) release. The present results show for the first time a dose-dependent increase in the phosphorylation of Ser2815 of RyR2 through the PKA-dependent activation of CaMKII and a predominant role of CaMKII-dependent phosphorylation of RyR2, over that of PKA-dependent phosphorylation, on SR-Ca(2+) release during betaAR stimulation.     

Regulation of sarcolemmal Ca2+ pump by endogenous protein phosphatases

Summary The function of several key sarcolemmal proteins is modulated through phosphorylation-dephosphorylation of serine/threonine residues. While the involvement of sarcolemma-associated protein kinases in the phosphorylation of these proteins has been established, the nature of the protein phosphatases controlling these proteins has not been investigated. Rat heart sarcolemma contains two protein phosphatase isozymes, protein phosphatase 1 and 2A, which are distinguished on the basis of their susceptibility of inhibitor 2. Both isozymes elute from a Bio Gel A-0.5 column in association with the highest molecular weight protein fraction. However, some protein phosphatase 1 activity elutes with a smaller molecular weight fraction of about 37000, suggesting that the native enzyme exists as a catalytic subunit in complex with an anchor protein. Inhibition of the protein phosphatases using standard inhibitors leads to a stimulation in both the rate and extent of³²P incorporation into isolated sarcolemma. Also affected by inhibition of protein phosphatase activity is the rate of ATP-dependent calcium uptake, which is stimulated following exposure to either inhibitor 2, a classical protein phosphatase 1 inhibitor, and microcystin, a protein phosphatase 1 and 2A inhibitor. The data suggest that the protein phosphatases regulate the dephosphorylation of sarcolemmal proteins Through this mechanism they serve as important modulators of the sarcolemmal Ca²⁺ pump.     

Beat-to-beat Ca(2+)-dependent regulation of sinoatrial nodal pacemaker cell rate and rhythm

Whether intracellular Ca²⁺ regulates sinoatrial node cell (SANC) action potential (AP) firing rate on a beat-to-beat basis is controversial. To directly test the hypothesis of beat-to-beat intracellular Ca²⁺ regulation of the rate and rhythm of SANC we loaded single isolated SANC with a caged Ca²⁺ buffer, NP-EGTA, and simultaneously recorded membrane potential and intracellular Ca²⁺. Prior to introduction of the caged Ca²⁺ buffer, spontaneous local Ca²⁺ releases (LCRs) during diastolic depolarization were tightly coupled to rhythmic APs (r² = 0.9). The buffer markedly prolonged the decay time (T₅₀) and moderately reduced the amplitude of the AP-induced Ca²⁺ transient and partially depleted the SR load, suppressed spontaneous diastolic LCRs and uncoupled them from AP generation, and caused AP firing to become markedly slower and dysrhythmic. When Ca²⁺ was acutely released from the caged compound by flash photolysis, intracellular Ca²⁺ dynamics were acutely restored and rhythmic APs resumed immediately at a normal rate. After a few rhythmic cycles, however, these effects of the flash waned as interference with Ca²⁺ dynamics by the caged buffer was reestablished. Our results directly support the hypothesis that intracellular Ca²⁺ regulates normal SANC automaticity on a beat-to-beat basis.     

Relationship between Ca2+-affinity and shielding of bulk water in the Ca2+-pump from molecular dynamics simulations

The sarcoplasmic reticulum Ca(2+)-ATPase transports two Ca(2+) per ATP hydrolyzed from the cytoplasm to the lumen against a large concentration gradient. During transport, the pump alters the affinity and accessibility for Ca(2+) by rearrangements of transmembrane helices. In this study, all-atom molecular dynamics simulations were performed for wild-type Ca(2+)-ATPase in the Ca(2+)-bound form and the Gln mutants of Glu771 and Glu908. Both of them contribute only one carboxyl oxygen to site I Ca(2+), but only Glu771Gln completely looses the Ca(2+)-binding ability. The simulations show that: (i) For Glu771Gln, but not Glu908Gln, coordination of Ca(2+) was critically disrupted. (ii) Coordination broke at site II first, although Glu771 and Glu908 only contribute to site I. (iii) A water molecule bound to site I Ca(2+) and hydrogen bonded to Glu771 in wild-type, drastically changed the coordination of Ca(2+) in the mutant. (iv) Water molecules flooded the binding sites from the lumenal side. (v) The side chain conformation of Ile775, located at the head of a hydrophobic cluster near the lumenal surface, appears critical for keeping out bulk water. Thus the simulations highlight the importance of the water molecule bound to site I Ca(2+) and point to a strong relationship between Ca(2+)-coordination and shielding of bulk water, providing insights into the mechanism of gating of ion pathways in cation pumps.     

Sarcolipin and phospholamban as regulators of cardiac sarcoplasmic reticulum Ca2+ ATPase

The cardiac sarcoplasmic reticulum calcium ATPase (SERCA2a) plays a critical role in maintaining the intracellular calcium homeostasis during cardiac contraction and relaxation. It has been well documented over the years that altered expression and activity of SERCA2a can lead to systolic and diastolic dysfunction. The activity of SERCA2a is regulated by two structurally similar proteins, phospholamban (PLB) and sarcolipin (SLN). Although, the relevance of PLB has been extensively studied over the years, the role SLN in cardiac physiology is an emerging field of study. This review focuses on the advances in the understanding of the regulation of SERCA2a by SLN and PLB. In particular, it highlights the similarities and differences between the two proteins and their roles in cardiac patho-physiology.     

Structural basis for the high Ca2+ affinity of the ubiquitous SERCA2b Ca2+ pump

Sarco(endo)plasmic reticulum Ca²⁺ ATPase (SERCA) Ca²⁺ transporters pump cytosolic Ca²⁺ into the endoplasmic reticulum, maintaining a Ca²⁺ gradient that controls vital cell functions ranging from proliferation to death. To meet the physiological demand of the cell, SERCA activity is regulated by adjusting the affinity for Ca²⁺ ions. Of all SERCA isoforms, the housekeeping SERCA2b isoform displays the highest Ca²⁺ affinity because of a unique C-terminal extension (2b-tail). Here, an extensive structure–function analysis of SERCA2b mutants and SERCA1a2b chimera revealed how the 2b-tail controls Ca²⁺ affinity. Its transmembrane (TM) segment (TM11) and luminal extension functionally cooperate and interact with TM7/TM10 and luminal loops of SERCA2b, respectively. This stabilizes the Ca²⁺-bound E1 conformation and alters Ca²⁺-transport kinetics, which provides the rationale for the higher apparent Ca²⁺ affinity. Based on our NMR structure of TM11 and guided by mutagenesis results, a structural model was developed for SERCA2b that supports the proposed 2b-tail mechanism and is reminiscent of the interaction between the α- and β-subunits of Na⁺,K⁺-ATPase. The 2b-tail interaction site may represent a novel target to increase the Ca²⁺ affinity of malfunctioning SERCA2a in the failing heart to improve contractility.     

Histamine-induced Ca(2+) signaling in human valvular myofibroblasts

In this study, we examined histamine-induced calcium signaling in cultured human valvular myofibroblasts (hVMFs), which are the most prominent interstitial cells in cardiac valves mediating valvular contraction, extracellular matrix secretion, and wound repair. Despite the functional importance of VMFs in cardiac valves, the cellular-signaling pathways, especially those mediated by Ca(2+), are still poorly understood. Using fluorescence imaging microscopy, we measured intracellular Ca(2+) ([Ca(2+)](i)) levels in fura-2-loaded hVMFs. Activation of H(1) receptors released Ca(2+) from one compartment of the endoplasmic reticulum (ER) of hVMFs, but did not induce Ca(2+) entry. This histamine-induced Ca(2+) release was oscillatory and dependent on Ca(2+) re-uptake into the ER by sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA). Application of the reversible SERCA blocker, cyclopiazonic acid (CPA), after depletion of the histamine-sensitive Ca(2+) store revealed the presence of a second, smaller histamine-insensitive Ca(2+) store in the ER. The Ca(2+) content ratio of the histamine-sensitive and histamine-insensitive Ca(2+) stores in the ER was found to be approximately 1.15:1. Another effect of CPA in hVMFs was the activation of store-operated Ca(2+) channels, as demonstrated by maintained [Ca(2+)](i) elevation as well as accelerated Mn(2+) entry. In conclusion, this study establishes for the first time an agonist-induced Ca(2+)-signaling pathway in hVMFs.     

异丙肾上腺素对心肌细胞内钙释放和肌浆网内钙容量的影响

目的 :心肌细胞膜上的 β-肾上腺素受体的激活对兴奋 -收缩耦联 （ECC）过程有重要的调节作用。本课题利用异丙肾上腺素 （ISO,1μmol/ L）激活 β-肾上腺素受体从而研究其对源自心肌细胞肌浆网的胞内钙释放 （ECC的重要环节 ）和肌浆网内钙容量的影响 ,进而分析钙释放与钙容量之间的关系。方法 :局部场刺激作用于成年大鼠心肌细胞 ,促使后者产生动作电位 ,进而诱发胞内钙瞬变 （ACT） ,由 ACT可估测胞内钙释放。肌浆网内钙容量则由咖啡因 （2 0 m mol/ L）诱发的钙瞬变 （CCT）估测。实验结果均由 Zeiss L SM- 5 10激光共聚焦显微镜系统记录。结果 :ISO作用下的 ACT峰值为 10 .2 9± 0 .35 （n=13）比正常情况下的 5 .74± 0 .2 7（n=18）高 （P<0 .0 1）。 ISO作用下的CCT峰值为 11.2 3± 0 .2 9（n=13）比正常情况下的 7.6 2± 0 .2 4 （n=18）高 （P<0 .0 1）。结论 :ISO可明显地提高心肌细胞内钙释放量和肌浆网内的钙容量。不管有无 ISO存在 ,胞内钙释放量总是只占肌浆网内钙容量的一部分。在正常情况下 ,心肌的钙释放量有较大的储备能力 ,且此储备可因β-肾上腺素受体的激活而动员。     

Targeted expression of the inositol 1,4,5-triphosphate receptor (IP3R) ligand-binding domain releases Ca2+ via endogenous IP3R channels

Virtually all functions of a cell are influenced by cytoplasmic [Ca(2+)] increases. Inositol 1,4,5-trisphosphate receptor (IP(3)R) channels, located in the endoplasmic reticulum (ER), release Ca(2+) in response to binding of the second messenger, IP(3).IP(3)Rs thus are part of the information chain interpreting external signals and transforming them into cytoplasmic Ca(2+) transients. IP(3)Rs function as tetramers, each unit comprising an N-terminal ligand-binding domain (LBD) and a C-terminal channel domain linked by a long regulatory region. It is not yet understood how the binding of IP(3) to the LBD regulates the gating properties of the channel. Here, we use the expression of IP(3) binding protein domains tethered to the surface of the endoplasmic reticulum (ER) to show that the all-helical domain of the IP(3)R LBD is capable of depleting the ER Ca(2+) pools by opening the endogenous IP(3)Rs, even without IP(3) binding. This effect requires the domain to be within 50 A of the ER membrane and is impaired by the presence of the N-terminal inhibitory segment on the LBD. These findings raise the possibility that the helical domain of the LBD functions as an effector module possibly interacting with the channel domain, thereby being part of the gating mechanisms by which the IP(3)-induced conformational change within the LBD regulates Ca(2+) release.     

Comparison of sarcoplasmic reticulum Ca2+-ATPase function in human,dog, rabbit,and mouse ventricular myocytes

It has been reported that sarcoplasmic reticulum (SR) Ca(2+) uptake is more rapid in rat than rabbit ventricular myocytes, but little information is available on the relative SR Ca(2+) uptake activity in others species, including humans. We induced Ca(2+) transients with a short caffeine pulse protocol (rapid solution switcher, 10 mM caffeine, 100 ms) in single ventricular myocytes voltage clamped (-80 mV) with pipettes containing 100 microM fluo-3 and nominal 0 Ca(2+), in 0 Na(+)(o)/0 Ca(2+)(o) solution to inhibit Na/Ca exchange. SR in non-paced human, dog, rabbit, and mouse ventricular myocytes could be readily loaded with Ca(2+) under our experimental conditions with a pipette [Ca(2+)] = 100 nM. Resting [Ca(2+)](i) was similar in four types of ventricular myocytes. Activation of the Ca(2+)-release channel with a 100-ms caffeine pulse produced a rise in [caffeine](i) to slightly above 2 mM, the threshold for caffeine activation of Ca(2+) release. This caused a similar initial rate of rise and peak [Ca(2+)](i) in the four types of ventricular myocytes. However, there were significant differences in the duration of the plateau (top 10%) [Ca(2+)](i) transients and the time constant of the [Ca(2+)](i) decline (reflecting activity of the SR Ca(2+)-ATPase), with values for human > dog > rabbit > mouse. In paced myocytes under physiologic conditions, SR Ca(2+) content was greater in mouse than in rabbit myocytes, while peak I(Ca,L) was smaller in mouse. These findings confirm substantial species difference in SR Ca(2+)-ATPase activity, and suggest that the smaller the animal and the more rapid the heart rate, greater the activity of the SR Ca(2+)-ATPase. In addition, it appears that substantial species differences exist in the degree of SR Ca(2+) loading and I(Ca,L) under physiologic conditions.     

Loss of beta-adrenoceptor response in myocytes overexpressing the Na+/Ca(2+)-exchanger

Increased Na+/Ca(2+)-exchanger (NCX) and altered beta-adrenoceptor (betaAR) responses are observed in failing human heart. To determine the possible interaction between these changes, we investigated the effect of NCX overexpression on responses to isoproterenol in adult rat ventricular myocytes. Responses to isoproterenol were largely mediated through the beta1AR in control myocytes. Adenovirally-mediated overexpression of NCX, at levels, which did not alter basal contraction of myocytes, markedly depressed the isoproterenol concentration-response curve. Responses to isoproterenol could be restored to normal by beta2AR blockade, suggesting a beta2AR-mediated inhibition of beta1AR signalling. Pertussis toxin normalised isoproterenol responses in NCX cells, indicating that beta2AR effects were mediated by Gi. Negative-inotropic effects of high concentrations of ICI 118,551, previously shown to be due to beta2AR-Gi coupling, were increased in NCX cells. We conclude that NCX upregulation can markedly alter the consequences of betaAR stimulation and that this may contribute to the alterations in betaAR response seen in failing human heart.     

A SERCA2 pump with an increased Ca2+ affinity can lead to severe cardiac hypertrophy, stress intolerance and reduced life span

Abnormal Ca(2+) cycling in the failing heart might be corrected by enhancing the activity of the cardiac Ca(2+) pump, the sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a (SERCA2a) isoform. This can be obtained by increasing the pump's affinity for Ca(2+) by suppressing phospholamban (PLB) activity, the in vivo inhibitor of SERCA2a. In SKO mice, gene-targeted replacement of SERCA2a by SERCA2b, a pump with a higher Ca(2+) affinity, results in cardiac hypertrophy and dysfunction. The stronger PLB inhibition on cardiac morphology and performance observed in SKO was investigated here in DKO mice, which were obtained by crossing SKO with PLB(-/-) mice. The affinity for Ca(2+) of SERCA2 was found to be further increased in these DKO mice. Relative to wild-type and SKO mice, DKO mice were much less spontaneously active and showed a reduced life span. The DKO mice also displayed a severe cardiac phenotype characterized by a more pronounced concentric hypertrophy, diastolic dysfunction and increased ventricular stiffness. Strikingly, beta-adrenergic or forced exercise stress induced acute heart failure and death in DKO mice. Therefore, the increased PLB inhibition represents a compensation for the imposed high Ca(2+)-affinity of SERCA2b in the SKO heart. Limiting SERCA2's affinity for Ca(2+) is physiologically important for normal cardiac function. An improved Ca(2+) transport in the sarcoplasmic reticulum may correct Ca(2+) mishandling in heart failure, but a SERCA pump with a much higher Ca(2+) affinity may be detrimental.     

From the Cover: Ryanodine receptor fragmentation and sarcoplasmic reticulum Ca2+ leak after one session of high-intensity interval exercise

High-intensity interval training (HIIT) is a time-efficient way of improving physical performance in healthy subjects and in patients with common chronic diseases, but less so in elite endurance athletes. The mechanisms underlying the effectiveness of HIIT are uncertain. Here, recreationally active human subjects performed highly demanding HIIT consisting of 30-s bouts of all-out cycling with 4-min rest in between bouts (≤3 min total exercise time). Skeletal muscle biopsies taken 24 h after the HIIT exercise showed an extensive fragmentation of the sarcoplasmic reticulum (SR) Ca²⁺ release channel, the ryanodine receptor type 1 (RyR1). The HIIT exercise also caused a prolonged force depression and triggered major changes in the expression of genes related to endurance exercise. Subsequent experiments on elite endurance athletes performing the same HIIT exercise showed no RyR1 fragmentation or prolonged changes in the expression of endurance-related genes. Finally, mechanistic experiments performed on isolated mouse muscles exposed to HIIT-mimicking stimulation showed reactive oxygen/nitrogen species (ROS)-dependent RyR1 fragmentation, calpain activation, increased SR Ca²⁺ leak at rest, and depressed force production due to impaired SR Ca²⁺ release upon stimulation. In conclusion, HIIT exercise induces a ROS-dependent RyR1 fragmentation in muscles of recreationally active subjects, and the resulting changes in muscle fiber Ca²⁺-handling trigger muscular adaptations. However, the same HIIT exercise does not cause RyR1 fragmentation in muscles of elite endurance athletes, which may explain why HIIT is less effective in this group.It is increasingly clear that regular physical exercise plays a key role in the general well-being, disease prevention, and longevity of humans. Impaired muscle function manifesting as muscle weakness and premature fatigue development are major health problems associated with the normal aging process as well as with numerous common diseases (1). Physical exercise has a fundamental role in preventing and/or reversing these muscle problems, and training also improves the general health status in numerous diseases (2–4). On the other side of the spectrum, excessive muscle use can induce prolonged force depressions, which may set the limit on training tolerance and performance of top athletes (5, 6).Recent studies imply a key role of the sarcoplasmic reticulum (SR) Ca²⁺ release channel, the ryanodine receptor 1 (RyR1), in the reduced muscle strength observed in numerous physiological conditions, such as after strenuous endurance training (6), in situations with prolonged stress (7), and in normal aging (8, 9). Defective RyR1 function is also implied in several pathological states, including generalized inflammatory disorders (10), heart failure (11), and inherited conditions such as malignant hyperthermia (12) and Duchenne muscular dystrophy (13). In many of the above conditions, there is a link between the impaired RyR1 function and modifications induced by reactive oxygen/nitrogen species (ROS) (6, 8, 10, 12, 13). Conversely, altered RyR1 function may also be beneficial by increasing the cytosolic free [Ca²⁺] ([Ca²⁺]_i) at rest, which can stimulate mitochondrial biogenesis and thereby increase fatigue resistance (14–16). Intriguingly, effective antioxidant treatment hampers beneficial adaptations triggered by endurance training (17–19), and this effect might be due to antioxidants preventing ROS-induced modifications of RyR1 (20).A high-intensity interval training (HIIT) session typically consists of a series of brief bursts of vigorous physical exercise separated by periods of rest or low-intensity exercise. A major asset of HIIT is that beneficial adaptations can be obtained with much shorter exercise duration than with traditional endurance training (21–25). HIIT has been shown to effectively stimulate mitochondrial biogenesis in skeletal muscle and increase endurance in untrained and recreationally active healthy subjects (22, 26), whereas positive effects in elite endurance athletes are less clear (21, 27, 28). Moreover, HIIT improves health and physical performance in various pathological conditions, including cardiovascular disease, obesity, and type 2 diabetes (29, 30). Thus, short bouts of vigorous physical exercise trigger intracellular signaling of large enough magnitude and duration to induce extensive beneficial adaptations in skeletal muscle. The initial signaling that triggers these adaptations is not known.In this study, we tested the hypothesis that a single session of HIIT induces ROS-dependent RyR1 modifications. These modifications might cause prolonged force depression due to impaired SR Ca²⁺ release during contractions. Conversely, they may also initiate beneficial muscular adaptations due to increased SR Ca²⁺ leak at rest.     

Presenilin 2 modulates endoplasmic reticulum (ER)-mitochondria interactions and Ca2+ cross-talk

Presenilin mutations are the main cause of familial Alzheimer's disease (FAD). Presenilins also play a key role in Ca(2+) homeostasis, and their FAD-linked mutants affect cellular Ca(2+) handling in several ways. We previously have demonstrated that FAD-linked presenilin 2 (PS2) mutants decrease the Ca(2+) content of the endoplasmic reticulum (ER) by inhibiting sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA) activity and increasing ER Ca(2+) leak. Here we focus on the effect of presenilins on mitochondrial Ca(2+) dynamics. By using genetically encoded Ca(2+) indicators specifically targeted to mitochondria (aequorin- and GFP-based probes) in SH-SY5Y cells and primary neuronal cultures, we show that overexpression or down-regulation of PS2, but not of presenilin 1 (PS1), modulates the Ca(2+) shuttling between ER and mitochondria, with its FAD mutants strongly favoring Ca(2+) transfer between the two organelles. This effect is not caused by a direct PS2 action on mitochondrial Ca(2+)-uptake machinery but rather by an increased physical interaction between ER and mitochondria that augments the frequency of Ca(2+) hot spots generated at the cytoplasmic surface of the outer mitochondrial membrane upon stimulation. This PS2 function adds further complexity to the multifaceted nature of presenilins and to their physiological role within the cell. We also discuss the importance of this additional effect of FAD-linked PS2 mutants for the understanding of FAD pathogenesis.     

Syntaxin 5 regulates the endoplasmic reticulum channel-release properties of polycystin-2

Polycystin-2 (PC2), the gene product of one of two genes mutated in dominant polycystic kidney disease, is a member of the transient receptor potential cation channel family and can function as intracellular calcium (Ca²⁺) release channel. We performed a yeast two-hybrid screen by using the NH₂ terminus of PC2 and identified syntaxin-5 (Stx5) as a putative interacting partner. Coimmunoprecipitation studies in cell lines and kidney tissues confirmed interaction of PC2 with Stx5 in vivo. In vitro binding assays showed that the interaction between Stx5 and PC2 is direct and defined the respective interaction domains as the t-SNARE region of Stx5 and amino acids 5 to 72 of PC2. Single channel studies showed that interaction with Stx5 specifically reduces PC2 channel activity. Epithelial cells overexpressing mutant PC2 that does not bind Stx5 had increased baseline cytosolic Ca²⁺ levels, decreased endoplasmic reticulum (ER) Ca²⁺ stores, and reduced Ca²⁺ release from ER stores in response to vasopressin stimulation. Cells lacking PC2 altogether had reduced cytosolic Ca²⁺ levels. Our data suggest that PC2 in the ER plays a role in cellular Ca²⁺ homeostasis and that Stx5 functions to inactivate PC2 and prevent leaking of Ca²⁺ from ER stores. Modulation of the PC2/Stx5 interaction may be a useful target for impacting dysregulated intracellular Ca²⁺ signaling associated with polycystic kidney disease.