In this investigation, skeletal muscle sarcoplasmic reticulum (SR) was purified from female Sprague Dawley rats (200-250 g). SR samples were subjected to two different biochemical glycogen-extraction protocols. The results suggest that both amylase and removal of EDTA (No-EDTA) from the homogenization and storage buffers reduced the amount of glycogen associated with the SR. Both of these treatments failed to impair SR calcium (Ca2+) handling when assayed under conditions where exogenous ATP was added and utilized for SR Ca2+ transport. In fact, these treatments seemed to cause a small increase in both SR Ca2+-uptake and release rates under these assay conditions. As expected, glycogen phosphorylase content was reduced as a result of glycogen extraction in the presence of amylase, however this was not the case for No-EDTA samples. Interestingly, many other proteins differed in content after glycogen extraction. These treatments resulted in a greater recovery of the sarco(endo)plasmic reticulum Ca2+ adenosine triphosphatase (SERCA) and a substantial loss of glycogen phosphorylase and glycogen debranching enzyme (AGL) in amylase-treated samples. Creatine kinase (CK) and pyruvate kinase (PK) contents were increased as a result of both glycogen-extraction conditions. It was imperative to consider these altered protein contents while analyzing the data and assessing the effects of glycogen extraction on SR Ca2+ handling. After normalizing to SERCA content, only No-EDTA samples had higher adenosine triphosphate (ATP)-supported SR Ca2+-uptake rates compared to control samples. For endogenously synthesized ATP-supported SR Ca2+-uptake experiments, normalizing data to protein content (either CK and SERCA or PK and SERCA) revealed that amylase-treated samples had lower SR Ca2+-uptake rates, compared to control samples. Although not significant, SR Ca2+-uptake rates for No-EDTA samples were also lower than control samples. These data suggest that changes in endogenously supported SR Ca2+-uptake due to glycogen extraction affected the source of ATP synthesis (either PK or CK), the effectiveness of energy utilization for Ca2+ transport (SERCA), or altered the metabolic channeling properties. / Ph. D.

Following contractile activity, fast twitch skeletal muscle exhibits increases in submaximal force known as potentiation. Although there is no consensus on the purpose of potentiation, it is known to enhance power during rapid dynamic contractions and counteract the early stages of peripheral fatigue. Potentiation is primarily attributed to phosphorylation of the myosin regulatory light chain (RLC) through a calcium-mediated process which results in increased calcium-sensitivity of crossbridge formation. However, there is a growing body of evidence showing that potentiation can be achieved in the absence of RLC phosphorylation, albeit to a lesser degree. A secondary characteristic of the potentiated contraction is an acceleration of relaxation properties, which could be teleologically beneficial to enhance the cycling rate of rapid motions (e.g. running). However, accelerated relaxation is inconsistent with elevations in calcium-sensitivity as this would tend to slow the time course and slow relaxation. Therefore there are multiple mechanisms involved in potentiation, some of which enhance crossbridge formation, and some of which enhance crossbridge detachment. A possible explanation for these events involves contraction-induced changes in the intracellular cytosolic calcium signal that triggers muscle contraction. For example, elevations in submaximal force could be achieved by increasing the amplitude of the calcium signal while enhanced relaxation speed could be achieved by a shorter duration of the calcium signal. Thus the main objective of this thesis was to investigate the contribution of changes in cytosolic Ca²⁺ to force potentiation. To achieve this objective, intact lumbrical muscles were extracted from the hind feet of C57BL/6 mice for use as the experimental model. The first study in this thesis examined cytosolic calcium signals during posttetanic potentiation using high (AM-fura-2 and AM-indo-1) and low (AM-furaptra) affinity calcium-sensitive fluorescent indicators to monitor resting and peak calcium respectively, both before and after a potentiating stimulation protocol of 2.5 s of 20 Hz stimulation at 37^oC. This protocol resulted in an immediate 17±3% increase in twitch force (n=10; P<0.05), though this potentiation dissipated quickly, lasting only 30 s. Resting cytosolic Ca²⁺ was also increased following the potentiating stimulus as indicated by increases of 11.1 ± 1.3% and 8.1 ± 1.3% in the fura-2 and indo-1 fluorescence ratios respectively. Like the force potentiation, these increases were short lived, lasting 20-30 s. No changes were detected in either the amplitude or kinetics of the Ca²⁺ transients following the potentiating stimulus. Western blotting analysis of the myosin heavy chain isoforms which determine the contractile phenotype of lumbrical muscle revealed predominance of fast type IIX fibres, while immunohistochemical analysis of proteins important for relaxation, namely parvalbumin, sarco-endoplasmic reticulum Ca²⁺ ATPase (SERCA) 1a and SERCA2a, revealed that the expression of these proteins in lumbrical moderated those found in the soleus (slow) and EDL (fast) archetypes. Surprisingly, despite the fast phenotype of the lumbrical, it exhibited low expression of the skeletal muscle isoform of myosin light chain kinase, the enzyme responsible for phosphorylating the myosin RLC, and high expression of myosin targeting phosphatase subunit 2, the enzyme responsible for dephosphorylating the myosin RLC. These data were corroborated by a complete lack of myosin RLC phosphorylation in either the rested or potentiated states. It was thus concluded that elevations in resting cytosolic calcium concentration, in the absence of changes in the intracellular calcium transient and RLC phosphorylation, can potentiate twitch force. The next objective of this thesis was to determine if there are changes in the cytosolic calcium transient during staircase potentiation, defined as a stepwise increase in twitch force during low frequency stimulation (<10 Hz). Staircase potentiation has been repeatedly demonstrated to exhibit more robust potentiation than posttetanic potentiation in the absence of RLC phosphorylation. It was hypothesized that while the calcium transient is not altered during posttetanic potentiation, it may be an important potentiating factor in staircase due to the lower rest intervals between successive contractions. The effects of temperature on the intracellular calcium transient during staircase potentiation were also examined as part of this investigation. Here, lumbricals were loaded with AM- furaptra and then subjected to stimulation at 8 Hz for 8.0 s to induce staircase potentiation at either 30 or 37^oC. This stimulation protocol resulted in a 26.8 ± 3.2 % increase in twitch force at 37^oC (P<0.05) and a 6.8 ± 1.9 % decrease in twitch force at 30^oC (P<0.05) at the 8 s mark. Both the peak amplitude and the calcium-time integral of the calcium transient decreased during the first 2.0 s of the protocol (P<0.05), however these decreases were greater at 30^oC than 37^oC (P<0.05 amplitude; P=0.09 area). While peak amplitude remained low throughout the duration of the protocol, the calcium-time integral began to increase after the 2 s time point (P<0.05), a change reflective of the progressive increases in the 50% decay time and full width at half maximum of the calcium transient (P<0.05). Regression analysis of raw furaptra fluorescence ratios revealed a progressive decline in the peak amplitude of the calcium transients throughout the protocol which was not present at 37^oC. The increases in the duration of the calcium transient were mirrored by increases in the half relaxation time of the twitch contractions at both 30 and 37^oC, which had initially been reduced by ~20 and 9 % at 30 and 37^oC during the first 2 s of the protocol. Therefore the degree of staircase potentiation depends, in part, on the magnitude of the decline in the amplitude and the degree of slowing of the cytosolic calcium transient. The declines in calcium transient amplitude noted above occurred simultaneously with increased rates of relaxation and abbreviated contraction times. To determine if there was a causal relationship between the reduced amplitude and the faster contractions, AM-furaptra-loaded lumbrical muscles were stimulated at 8 Hz for 2 s in the presence and absence of caffeine, an agonist of the calcium release channel. Caffeine treatment attenuated the decline of the calcium transient amplitude (P<0.05), and was associated with greater potentiation at 37^oC (P<0.05), and attenuated force loss at 30^oC (P<0.05). Despite the increases in calcium and force, the relaxation times and rates of relaxation exhibited a greater acceleration following caffeine treatment (P<0.05). Therefore the relaxation-enhancing factor during potentiated twitches cannot be attributed to the calcium transient, and must be localized to changes on the myofilament. The case for inorganic phosphate as the effector is made. Similar to the findings of the posttetanic potentiation study, the resting cytosolic calcium concentration was elevated during staircase potentiation, as revealed by fura-2 ratio signals. The largest increase occurring immediately following the first twitch of the protocol. This coincided with the largest increases in force potentiation at both 30 and 37^oC. This finding is in accordance with the initial conclusion that elevations in resting calcium can enhance twitch force and contribute to potentiation, though the mechanism of action is unclear. One possibility is that increases in resting calcium, sub-threshold for force production, can enhance the number of attached but non-force producing crossbridges, thereby accelerating the transition of crossbridges to force-producing states upon calcium-release following stimulation. To test this hypothesis, the resting stiffness, a measure of crossbridge attachment, of lumbrical muscles was examined before and after a potentiating stimulus of 20 Hz 2.5 s. Resting stiffness was assessed using sinusoidal length oscillations, ~0.5 nm per half sarcomere in amplitude and ranging in frequency from 10-200 Hz. Subsequent analysis revealed decreases in the elastic stiffness (P<0.05) that lasted for ~20 s which were greater in magnitude (P<0.05) than increases in viscous stiffness which only lasted for ~5 s. This finding is consistent with the disappearance of short range elastic component (SREC) upon stretch or muscle activation which is commonly attributed to a population of stable, bound crossbridges in resting muscle. Subsequent analysis using imposed length changes to eliminate the SREC prior to contraction had no effect on the amplitude or duration of a subsequent twitch or tetanic contraction, and the changes in elastic and viscous stiffness of resting muscle were identical whether SREC was ablated by a contraction or imposed length change. Therefore it appears that potentiation occurs without an associated increase in bound crossbridges at rest, and may actually occur with fewer bound crossbridges at rest than the unpotentiated state. The lack of effect may be related to the relaxation-enhancing factor discussed above, and be an important feature of skeletal muscle serving to protect against damage via an involuntary eccentric contraction. This thesis describes potentiation as a complex and important biological function which is the sum of factors that serve to enhance and oppose force production.

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This thesis develops biophysically-based data-driven mathematical models of intracellular calciumdynamics in ventricularmyocytes for both normal and genetically modified mouse hearts, based on species- and temperature-consistent experimental data. The models were subsequently applied to quantitatively examine the changes in calcium dynamics in mice with cardiomyocyte-specific knockout (KO) of the cardiac sarco/endoplasmic reticulum ATPase (SERCA2) gene, to determine the contributing mechanisms which underlie the ultimate development of heart failure in these animals. In Chapter 1, with emphasis on calcium dynamics and calcium regulation in heart failure, an overview of cardiac electrophysiology, excitation-contraction coupling and mathematical models of cardiac electrophysiology is provided. In Chapter 2, models of calcium dynamics in the ventricular myocytes from the C57BL/6 mouse heart at a physiological temperature is developed and validated based on species- and temperature-consistent measurements. In Chapter 3, the C57BL/6 model framework is re-parameterised to experimental data from the control and SERCA2 KO mice at 4 weeks after gene deletion. The models are then used to quantitatively characterise changes in calcium dynamics in the KO animals and the role of the compensatory mechanisms. In Chapter 4, the model framework is extended to include differential distributions of ion channels in the sarcolemma and the calcium dynamics in the sub-sarcolemmal space, with parameters in these sub-components fitted to experimentally measured calcium dynamics from the control and KO cardiomyocytes at 7-week after gene deletion. Finally in Chapter 5, conclusions are drawn, the limitations of this study are discussed, and the future extensions to this study are described.

Orientador: Humberto Santo Neto / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-20T21:05:12Z (GMT). No. of bitstreams: 1 Ferretti_Renato_D.pdf: 15919724 bytes, checksum: 2e3e4ba8b562aaaaf3dde85b2e810561 (MD5) Previous issue date: 2012 / Resumo: Os músculos intrínsecos da laringe (MIL) são protegidos da mionecrose em camundongos mdx, modelo da distrofia muscular de Duchenne (DMD). A DMD caracteriza-se pela mionecrose, causada pela ausência da proteína distrofina (DIS). A DIS se une a um complexo glicoproteínas (CDG), que auxilia na estabilidade da fibra e interage com proteínas reguladoras do Ca2+. O sarcolema instável dado pela ausência da DIS, diminuição de proteínas do CDG, desregulação de canais de Ca2+ e leva ao influxo de Ca2+ exacerbado, associado à diminuição da homeostase do Ca2+ intracelular e capacidade do estresse oxidativa, resulta na mionecrose. A utrofina (Utrn), semelhante à proteína DIS, se conecta às proteínas do CDG, parece ter papel na agregação dos receptores de acetilcolina (AChRs) nas junções neuromusculares (JNM) e auxiliaria na proteção da mionecrose. Neste trabalho examinamos os níveis das proteínas do CDG (beta-distroglicana [b-DG], beta-sarcoglicana [b-SG] e alfa-sintrofina [a-SIN]), da Utrn, de canais de estoque do Ca2+ (SOCE; proteínas Orai e STIM1) e proteína relacionada ao estresse oxidativo na mitocôndria (PGC-1a) em MIL distróficos. Além disso, analisamos o padrão de distribuição da Utrn e AChRs nas JNM de MIL distróficos. A análise da b-DG, b-SG, a- SIN e da Utrn pela técnica de imunohistoquímica e western blotting, mostrou que nos MIL distróficos os níveis da b-DG e da Utrn são normais em MIL adultos comparados com controle. Com envelhecimento, os níveis de proteínas do CDG e Utrn são alterados em MIL distróficos e parece não estar relacionado a proteção da mionecrose. Com emprego de técnicas moleculares e bioquímicas para análise de canais de estoque do Ca2+ (Orai e STIM1) e proteína do estress oxidativo na mitocôndria (PGC-1a), foram observados níveis aumentados de STIM1, com similar aumento da proteína calmodulina (CaM), bem como aumento da expressão do PGC-1a em MIL distróficos. Músculos acometidos distróficos apresentaram diminuição na expressão destas proteínas. Utilizando imunohistoquimica e microscopia confocal, foi observado que o padrão de distribuição da Utrn e dos AChRs não se altera nas JNM de MIL mdx. Em fibras regeneradas tanto o padrão de distribuição da Utrn quanto dos AChRs mostraram-se alterados. Pode-se concluir que os MIL de camundongos mdx apresentam manutenção dos níveis normais de proteínas do CDG e da Utrn, bem como o aumento de STIM1, CaM e PGC-1a, poderiam auxiliar, pelo menos em parte, à proteção da mionecrose / Abstract: The intrinsic laryngeal muscles (ILM) are protected from myonecrosis in the mdx mouse model of Duchenne muscular dystrophy (DMD). The DMD is characterized by myonecrosis, resulting from the absence of dystrophin protein. Dystrophin links the cytoskeleton to a complex of glycoprotein, the dystrophin-glycoprotein complex (DGC), which interacts with Ca2+-dependent channels for signaling and stability of the muscle membrane. In the absence of dystrophin, the sarcolemma becomes instable due to a decrease in the DGC level, deregulation of Ca2+-dependent channels, which increase Ca2+ influx, decreased Ca2+-handling and oxidative stress capacity, that result in muscle fiber necrosis. Utrophin (Utrn), similarly to the dystrophin, conects to DGC proteins, assembles the acethylcoline receptors (AChRs) at the neuromuscular junctions (NMJ) and may play a role in dystrophic muscle sparing. In this study, we examined the levels of DGC proteins (beta-dystroglycan [b-DG], beta-sarcoglycana [b-SG], alpha-syntrophin [a-SIN]), Utrn, store-operated Ca2+ channels (SOCE; STIM1 and Orai1 proteins), and mitochondrial oxidative protein (PGC-1a) in dystrophic ILM. We also examined the distribution of Utrn and AChRs in the dystrophic ILM NMJ. Immunohistochemistry and western blotting analyses of b-DG, b-SG and a-SIN, and the Utrn showed normal levels in adult dystrophic ILM compared with adult control. In aged mice, a dramatic decrease in DGC levels was observed in all dystrophic muscles compared with control. There are age-related alterations in DGC in the ILM of mdx mice, regardless of their protection against the lack of dystrophin. Using biochemical and molecular techniques to analyze SOCE proteins and mitochondrial oxidative protein (PGC-1a), we observed increased levels of STIM1, associated with increased level of calmodulin (CaM), and increased level of PGC-1a in dystrophic ILM. Dystrophic affected muscles have decreased levels of those proteins. Using molecular and biochemical methods we observed that Utrn and AChRs are fragmented only in affected muscle fibers and remaining unchanged in dystrophic MIL. We conclude that in adult dystrophic MIL the rescue of the DGC, increased levels of STIM1, CaM and PGC-1a, may be associated, at least in part, with their protection against myonecrosis / Doutorado / Anatomia / Doutor em Biologia Celular e Estrutural

Omega 3 poly unsaturated fatty acids (n-3 PUFA) have been shown to protect against sudden cardiac death following myocardial infarction and reduce the risk of ventricular arrhythmias in patients with heart failure. At the inception of this study, there was one clinical study that reported n-3 PUFA supplementation reduced the risk of atrial fibrillation (AF) following CABG. As AF is a very common arrhythmia and as there are no safe and effective means of preventing AF, we designed this study to further validate the findings of the previous study in a more robust study design. In addition, this study also aimed to evaluate the cellular changes that underpin the beneficial anti-arrhythmic effect of n-3 PUFA.The outcome of this study shows that n-3 PUFA does not reduce the risk of AF following CABG. However, short term supplementation with n-3 PUFA reliably increases the membrane incorporation in phospholipids and results in alteration in the expression levels of cardiac calcium handling proteins phospholamban and ryanodine receptors. In addition, such incorporation in animal (rat) ventricular myocytes leads to changes in the rate of decay of the systolic calcium transient and an increase in the amplitude of the caffeine induced calcium transient thereby indicating a greater activity of SERCA. These findings needs further evaluation but is clearly interesting as the clinical situations where n-3 PUFA have been shown to be anti-arrhythmic are situations where cellular calcium overload is the main mechanism of arrhythmogenesis.

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Orientador: Antonio Carlos Cicogna / Resumo: Introdução: Diversos modelos experimentais têm avaliado o processo de remodelação cardíaca (RC); dentre eles, destaca-se a indução à estenose aórtica supravalvar (EAo). Os mecanismos fisiopatológicos responsáveis pela depressão da função cardíaca incluem alterações no trânsito de cálcio (Ca2+) e em suas proteínas regulatórias. O treinamento físico (TF) tem sido utilizado na terapêutica das cardiopatias. Na patologia cardíaca por sobrecarga pressórica, o TF restaura, total ou parcialmente, a atividade e/ou expressão das proteínas regulatórias do trânsito de Ca2+, otimizando o fluxo de Ca2+ intracelular e atenuando o prejuízo funcional cardíaco. Objetivo: Analisar a participação do trânsito de Ca2+ e suas proteínas reguladoras na melhoria da função cardíaca de ratos com EAo e disfunção ventricular pelo TF. Material e Métodos: Ratos Wistar machos (70-90 g), submetidos à cirurgia de EAo, foram divididos em dois grupos: controle operado (Sham) e EAo. Após 18 semanas da cirurgia, foi analisada função cardíaca para redistribuição dos grupos: não expostos ao TF (Sham, n= 36 e EAo, n= 29) e treinados (ShamTF, n= 33 e EAoTF, n= 32) durante 10 semanas. O treinamento físico aeróbio (TFa) em esteira foi realizado com velocidade equivalente ao limiar de lactato, obtida durante os testes de esforço (inicial, 4a e 7a semanas e final). A RC foi avaliada por ecocardiografia, músculo papilar e cardiomiócito isolados e macroscopia post mortem. O trânsito de cálcio miocárdico foi analisado pela e... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction: Several experimental models have been proposed for the study of cardiac remodeling (CR); among them, the induction of supravalvular aortic stenosis (AoS). The pathophysiological mechanisms responsible for the cardiac function depression include changes in calcium (Ca2+) and its regulatory proteins. Exercise training (ET) has been used in the management of cardiopathies. In cardiac pathology due to pressure overload, ET completely or partially restores the activity and/or expression of regulatory proteins of Ca2+ handling, optimizing intracellular Ca 2+ flow and attenuating cardiac functional impairment. Objective: To analyze the participation of Ca2+ handling and its regulatory proteins in the improvement of the cardiac function of rats with aortic stenosis and ventricular dysfunction by ET. Material and Methods: Male Wistar rats (70-90 g) submitted to supravalvular aortic stenosis (AoS) were divided into two groups: operated control (Sham) and aortic stenosis (AoS). After 18 weeks of the surgical procedure, cardiac function analysis was performed for redistribution of the groups: non-exposed to exercise training (Sham, n = 36 and AoS, n = 29) and trained (ShamET, n = 33 and AoSET, n = 32) for 10 weeks. The treadmill exercise training was performed with a velocity equivalent to the lactate threshold, obtained during effort tests (initial, 4th and 7th weeks, and final). CR was evaluated by echocardiography, papillary muscle and cardiomyocyte isolated and postmort... (Complete abstract click electronic access below) / Doutor

Abstract Cardiac and skeletal muscle cell contraction is a result of excitation-contraction coupling (ECC), where an electrical signal leads to a rise in intracellular calcium levels and contraction. This process is carefully regulated to meet physiological demand and heavily dependent on an adequate energy supply. Disturbed ECC can have severe consequences on muscle cell function and underlies many cardiac and skeletal muscle pathologies. Cell stress, changing intracellular Ca2+ concentrations, and calcium signal dynamics can all play a role in the transcriptional regulation of genes involved in myocyte Ca2+-handling. In this thesis project, the transcriptional control of ECC was studied in skeletal and cardiac myocytes. Skeletal myocyte calsequestrin (CASQ1) was downregulated in a mouse model of mitochondrial myopathy and it contributed to the decreased SR Ca2+ load and impaired Ca2+ handling in Tfam-/- skeletal myocytes. In cultured neonatal cardiomyocytes, mitochondrial uncoupler FCCP-induced mitochondrial dysfunction led to downregulation of cardiac calsequestrin (CASQ2) and similarly impaired Ca2+ handling. Whereas there was no increase in reactive oxygen species (ROS) levels in Tfam-/- myocytes, cultured cells exposed to FCCP did display increased ROS, an effect that was counteracted by coexposure with the ROS scavenger (NAC). NAC attenuated FCCP-induced CASQ2 downregulation and restored Ca2+ handling. Therefore, mitochondrial dysfunction led to CASQ1/2 downregulation and impaired Ca2+ handling in these two cell types, but by different mechanisms. This project also looked at the role of Ca2+ dynamics on the transcriptional regulation of Ca2+ handling genes. Increased intracellular Ca2+ levels and &#946;-adrenergic stimulation of cardiomyocytes activate Ca2+-calmodulin kinase II (CaMKII) and can trigger hypertrophic remodeling. It was found that CaMKII downregulated expression of the L-type Ca2+ channel &#945;1c-subunit (Cacna1c) in cultured cardiomyocytes. Analysis of the Cacna1c promoter revealed that the transcriptional repressor DREAM bound to a putative downstream regulatory element. The results shed light on the complex interplay between muscle cell energetics and transcriptional regulation of SR Ca2+ handling proteins. A unique pathway for Cacna1c transcriptional regulation by CaMKII and DREAM was also described. / Tiivistelmä Sydän- ja luustolihassolujen supistuminen on seurausta ärsytys-supistuskytkennästä (ECC), jossa sähköinen ärsytys kohottaa solunsisäistä kalsiumpitoisuutta ja aiheuttaa supistuksen. Tätä säädellään tarkasti fysiologisen tarpeen mukaan, ja se riippuu riittävästä energian saannista. Häiriintynyt ECC voi aiheuttaa vakavia seurauksia lihassolujen toiminnalle, ja se on mukana monien sydän- ja luustolihasten sairauksien synnyssä. Tässä tutkimuksessa ECC:n transkriptionaalista säätelyä tutkittiin luustolihasten ja sydämen lihassoluissa. Luustolihassolujen kalsekvestriinin (CASQ1) väheneminen pienensi SR:n Ca2+-määrää mitokondrioiden myopatian hiirimallissa ja heikensi Ca2+-tasapainon ylläpitoa Tfam-/--luustolihassoluissa. Viljellyissä vastasyntyneiden kammio-sydänlihassoluissa mitokondrio-irtikytkijän FCCP:n aiheuttama mitokondrioiden toimintahäiriö johti sydämen kalsekvestriinin (CASQ2) vähenemiseen ja heikensi samalla tavalla Ca2+-tasapainon ylläpitoa. Vaikka Tfam-/--myosyyteissä reaktiivisten happilajien (ROS) tasot eivät olleet koholla, FCCP:lle altistetuissa viljellyissä soluissa ROS kuitenkin lisääntyi. Vaikutusta esti ROS-puhdistaja NAC, joka heikensi FCCP:n aiheuttamaa CASQ2:n laskua ja palautti Ca2+-säätelyn normaaliksi. Mitokondrioiden toimintahäiriö siis johti CASQ1/2:n vähenemiseen ja Ca2+-säätelyn heikentymiseen molemmissa solutyypeissä, mutta eri mekanismeilla. Tässä tutkimuksessa tarkasteltiin myös Ca2+-dynamiikan osuutta Ca2+-tasapainoon osallistuvien geenien transkription säätelyssä. Lisääntynyt solunsisäinen Ca2+-taso ja sydänlihassolujen &#946;-adrenerginen stimulointi aktivoivat Ca2+-kalmoduliinikinaasi II:n (CaMKII), ja ne voivat laukaista sydämen hypertrofisen uudelleenmuovautumisen. Havaittiin, että CaMKII vähensi L-tyypin Ca2+-kanavan a1c-alayksikön (Cacna1c) ilmentymistä viljellyissä sydänlihassoluissa. Promoottorianalyysi osoitti tämän johtuvan transkription repressorin DREAM:n sitoutumisesta oletettuun DRE:hen (alavirrassa sijaitseva säätelyelementti). Nämä tulokset tuovat uutta tietoa lihassolujen energiatalouden ja SR:n Ca2+:n vaikuttavien proteiinien transkription säätelyn vuorovaikutuksesta. Lisäksi havaittiin ainutlaatuinen Cacna1c-transkription säätelyn reitti, johon osallistuvat CaMKII ja DREAM.