AAV-Mediated Knock-Down of HRC Exacerbates Transverse Aorta Constriction-Induced Heart Failure

Background Histidine-rich calcium binding protein (HRC) is located in the lumen of sarcoplasmic reticulum (SR) that binds to both triadin (TRN) and SERCA affecting Ca2+ cycling in the SR. Chronic overexpression of HRC that may disrupt intracellular Ca2+ homeostasis is implicated in pathogenesis of cardiac hypertrophy. Ablation of HRC showed relatively normal phenotypes under basal condition, but exhibited a significantly increased susceptibility to isoproterenol-induced cardiac hypertrophy. In the present study, we characterized the functions of HRC related to Ca2+ cycling and pathogenesis of cardiac hypertrophy using the in vitro siRNA- and the in vivo adeno-associated virus (AAV)-mediated HRC knock-down (KD) systems, respectively. Methodology/Principal Findings AAV-mediated HRC-KD system was used with or without C57BL/6 mouse model of transverse aortic constriction-induced failing heart (TAC-FH) to examine whether HRC-KD could enhance cardiac function in failing heart (FH). Initially we expected that HRC-KD could elicit cardiac functional recovery in failing heart (FH), since predesigned siRNA-mediated HRC-KD enhanced Ca2+ cycling and increased activities of RyR2 and SERCA2 without change in SR Ca2+ load in neonatal rat ventricular cells (NRVCs) and HL-1 cells. However, AAV9-mediated HRC-KD in TAC-FH was associated with decreased fractional shortening and increased cardiac fibrosis compared with control. We found that phospho-RyR2, phospho-CaMKII, phospho-p38 MAPK, and phospho-PLB were significantly upregulated by HRC-KD in TAC-FH. A significantly increased level of cleaved caspase-3, a cardiac cell death marker was also found, consistent with the result of TUNEL assay. Conclusions/Significance Increased Ca2+ leak and cytosolic Ca2+ concentration due to a partial KD of HRC could enhance activity of CaMKII and phosphorylation of p38 MAPK, causing the mitochondrial death pathway observed in TAC-FH. Our results present evidence that down-regulation of HRC could deteriorate cardiac function in TAC-FH through perturbed SR-mediated Ca2+ cycling.


Introduction
The histidine-rich calcium binding protein (HRC), located in the luminal region of sarcoplasmic reticulum (SR), is a low-affinity and high-capacity Ca 2+ -binding protein [1,2,3]. The histidine-and glutamic acid-rich repeat region of HRC binds to the KEKE motif of the luminal region of triadin (TRN) [4], the site for binding to both calsequestrin (CSQ) [5,6] and ryanodine receptor (RyR) [7]. The same region of HRC also interacts with the N-terminal cation transporter domain of SR Ca 2+ -ATPase (SERCA) in a Ca 2+ concentration-dependent way [8]. However, the physiological importance of the multi-protein interactions between HRC and other proteins in the SR has remained to be clarified.
We have previously reported that HRC overexpression increased SR Ca 2+ load both in neonatal and adult rat cardiomyo-cytes [9]. In addition, adenovirus-mediated HRC overexpression in adult rat cardiomyocytes increased time to reach 50% relaxation (T 50 ) and time constant of decay, and decreased peak amplitude of Ca 2+ -induced Ca 2+ release, and fractional shortening [10]. Overexpression of HRC in transgenic mice resulted in impaired SR Ca 2+ uptake rates and depressed cardiomyocyte Ca 2+ transient decay, without significant changes in Ca 2+ transient amplitude or SR Ca 2+ load, indicating an inhibitory role of HRC for SERCA activity [11]. Furthermore, HRC transgenic mice expressed hypertrophic phenotypes developing increased heart weight/body weight ratio (HW/BW) and induction of fetal gene expression of atrial natriuretic factor (ANF) and b-myosin heavy chain (b-MHC) [11]. HRC knock-out (KO) mice showed relatively normal phenotypes under no stressful conditions, but exhibited a significantly increased susceptibility to isoproterenol Exacerbated Heart Failure in HRC-KD Heart PLOS ONE | www.plosone.org (ISO)-induced cardiac hypertrophy suggesting a regulatory role of HRC in the cardiac remodeling [12]. Collectively, HRC may be an important Ca 2+ cycling regulator in SR of which expression could be associated with pathogenesis of the heart. However, the precise mechanism of HRC mediated inhibition of Ca 2+ cycling and the long term cardiac remodeling has remained to be clarified.
The present study was designed on the basis of the hypothesis that HRC knock-down (KD) enhances Ca 2+ cycling and cardiac function through the increased activity of SERCA2 and RyR2. Thus, we used synthetic siRNA oligonucleotides and adenoassociated virus (AAV) to knock-down HRC expression in vitro (for short term effect) and in vivo (for chronic effect), respectively. HRC-KD in neonatal rat ventricular cells (NRVCs) or HL-1 cells showed enhanced Ca 2+ cycling, but the resting Ca 2+ concentration was increased due possibly to Ca 2+ leak through the activated RyR2. HRC-KD using AAV9-shHRC resulted in more decreased cardiac function, and increased cardiac fibrosis and apoptosis causing more severe heart failure in mice under pressure-overload by transverse aortic constriction (TAC). Our concomitant biochemical study showed that the increased Ca 2+ -leak and elevated cytosolic Ca 2+ due to HRC-KD could enhance phosphorylation of CaMKII -p38 MAPK pathway resulting in the increased apoptosis and heart failure. Collectively, the present study suggests that HRC is an important regulator of Ca 2+ homeostasis which is essential for normal functions of the heart.

Successful (KD) of HRC in NRVCs
The chronic HRC overexpression or KO studies presented higher expression of TRN protein [10,12]. The phenotypic changes occurring in response to overexpression or ablation of HRC could be associated with an adaptive developmental or compensatory response system-wide [13]. Therefore, in this study, an acute partial KD of HRC was performed in NRVCs using siRNA oligonucleotides targeted to HRC to evaluate the effects of decreased HRC expression on Ca 2+ cycling without possible change in protein expression related to Ca 2+ cycling. The siRNA oligonucleotides were successfully transfected to NRVCs (the calculated transfection efficiency was more than 99%) ( Figure 1A) and the cells showed a 70% reduction of HRC protein expression, where was no noticeable change of protein expression in RyR2, DHPR, NCX, SERCA2, CSQ, CaMKII, or TRN ( Figure 1). We also conducted the above experiments using HL-1 cells originally derived from mouse atrial cells. The results are similar between the different cell types ( Figure S1).
We also measured 20 mM caffeine-induced Ca 2+ 34.0) could be due to fact that Ca 2+ release was already activated under ISO condition in siNC cells. HL-1 cells showed no significant change of caffeine-induced Ca 2+ transients, but significantly enhanced fractional Ca 2+ release under basal condition, similar to NRVCs ( Figure S2C).  . Electrical-and caffeine-induced Ca 2+ transients in NRVCs. After 48 h of transfection with siNC or siHRC oligonucleotide, the NRVCs were treated with Fura 2-AM, and Ca 2+ transients were measured at 1-Hz electrical stimulation or 40 mM caffeine application in Tyrode solution to measure sarcoplasmic reticulum (SR) Ca 2+ load using IonOptix. A: Typical records of electrical-and caffeine-induced Ca 2+ transients in siNC and siHRC oligonucleotide treated NRVCs. B: Significantly changed parameters of Ca 2+ transients after HRC-KD. C: Left, the SR Ca 2+ load was not significantly different between siNC and siHRC oligo-transfected NRVCs Right, the fractional Ca 2+ release was significantly increased in HRC-KD NRVCs (siHRC). 16 [17,18]. Our ryanodine binding result suggests that HRC-KD Figure 3. Electrical-and caffeine-induced Ca 2+ transients in NRVCs treated with ISO. After 48 h of transfection with siNC or siHRC oligonucleotide, the NRVCs were treated with Fura 2-AM, Ca 2+ transients were measured at 1-Hz electrical stimulation at 1 mmol/L isoproterenol (ISO) using IonOptix. A: Typical records of Ca 2+ transients at 1 mmol/L ISO in siNC and siHRC oligonucleotide treated NRVCs. B: Significantly changed parameters of Ca 2+ transients after HRC-KD. Baseline, resting cytosolic Ca 2+ concentration; peak amplitude, the amount of Ca 2+ released from SR; T 50 , time to 50% baseline fluorescence; fractional Ca 2+ release, depolarization-induced Ca 2+ release/caffeine-induced Ca 2+ release (*P,0.05). doi:10.1371/journal.pone.0043282.g003 increases the open-state of RyR2 without noticeable change of ryanodine binding affinity.

AAV-mediated KD of HRC could Exacerbate the Heart Function in TAC-FH
We first attempted to use AAV serotype 9 for cardiac-specific gene transfer and tagged DsRed as a fluorescent infection marker to investigate whether KD of HRC could improve the heart function in TAC-FH model, since HRC-KD in NRVCs and HL-1 cells showed enhanced Ca 2+ cycling ( Figure 2 and Figure S3). However, KD of HRC was failed with the recombinant condition, due possibly to the inhibition by the fluorescent molecule, DsRed (19). Removal of DsRed in the construct significantly improved the HRC-KD efficiency ( Figure 5A). Since DsRed was removed for enhancing KD efficiency, we used AAV-GFP system instead to examine the transduction efficiency of AAV system indirectly ( Figure S4). The calculated transfection efficiency was approximately 30%. After 11 weeks of TAC operation, heart weight over body weight (HW/BW) ratio and heart weight over tibia length (HW/TL) ratio were significantly increased in both shNC and shHRC mice. However, there were no statistically significant differences in HW/BW and HW/TL between shNC and shHRC mice ( Figures 5B and 5C), indicating that heart failure was successfully induced by TAC in both cases. The results of KD showed that the expression of HRC was reduced 20% in the shHRC sham (n = 4) compared with shNC sham (n = 4), and 36% in the shHRC failure (n = 7) compared with shNC failure (n = 7) in vivo ( Figure 5E). It is interesting to note that the expression level of HRC was significantly decreased by TAC regardless of the animal models (shNC and shHRC), suggesting that HRC expression is associated with pathogenesis of the heart.
The partially HRC-KD mice and age-matched control samples in sham or TAC were subjected to echocardiography. The results showed that HRC-KD led to further reduction in fractional shortening (FS) and further increase in dilation of the heart at 11 weeks post operation in the failure group ( Figures 6A and 6B). HRC-KD also showed a further decrease of posterior wall thickness (PWT) and septal wall thickness (SWT) in the heart at 11 weeks post operation ( Table 1), indicative of severe heart failure in HRC-KD hearts. However, there was no significant difference in echocardiographic parameters between shNC sham and shHRC sham. We further performed trichrome staining to determine the degree of fibrosis in the different heart samples. There were no visible signs of fibrosis in the shNC sham and the shHRC sham, but the shHRC failure group showed more severe fibrosis compared with shNC failure group ( Figure 6C). These results suggest that HRC-KD alone did not induce cardiac dysfunction under normal conditions, but it could worsen cardiac dysfunction in heart failure conditions.

AAV-mediated KD of HRC Increased Cardiac Cell Death
It has been reported that apoptosis is the critical mechanism present in hypertensive heart disease [20], and cardiac apoptosis leads to decreased numbers of cardiomyocytes, with dead cells being replaced by fibrous tissues [21]. Since there was severe fibrosis in the shHRC failure group ( Figure 6C), we examined cardiac apoptosis by the TUNEL assay using paraffin-embedded heart tissues. TUNEL-positive cardiomyocyte nuclei were most abundant in shHRC failure hearts (n = 5) compared with shHRC sham (n = 5), shNC failure (n = 4) and shNC sham (n = 4) ( Figure 7A). Then, we checked the expression levels of proteins related to cell death. The largest expression of cleaved caspase-3, an apoptosis marker protein, was most abundant and Bax/Bcl-2 ratio was also increased in shHRC failure hearts ( Figure 7B), indicating that there was significantly increased cardiac apoptosis in the shHRC failure group causing the severe cardiac fibrosis. Thus, we further investigated the signaling mechanism of HRC-KD induced cardiac apoptosis and severe fibrosis in failing hearts.

AAV-mediated KD of HRC Activates CaMKII -p38 MAPK Signaling Pathway
CaMKII, a common intermediate of various death signalinduced apoptotic pathways in cardiac cells [22], has been highly associated with the transition from pressure overload-induced cardiac hypertrophy to heart failure in mice [23,24,25,26]. We investigated whether HRC-KD could induce CaMKII-mediated cardiac apoptosis by examining phosphorylation of CaMKII and its substrates, since HRC-KD increased cytosolic Ca 2+ concentration in NRVCs and HL-1 cells (Figure 2 and Figure S2). The results showed that phosphorylation of CaMKII increased 2.8 fold in shHRC failure, but increased only 1.2 fold in shHRC sham hearts. In addition, phosphorylation levels of phospholamban (PLB), RyR2, and p38 MAPK were also significantly increased, although total protein expressions of PLB, RyR2, CaMKII and p38 MAPK were not significantly changed in shHRC failure ( Figure 8). Phosphorylation of RyR2 and PLB may affect SR Ca 2+ cycling, possibly resulting in Ca 2+ leak [27,28,29]. The p38 MAPK pathway, downstream of CaMKII [30], was also significantly activated. However, the expression levels of other Ca 2+ cycling proteins were not changed, except for SERCA2a, which was down-regulated approximately 30%. Therefore, HRC-KD could further affect the mitochondrial death pathway by causing an imbalance between the expression of Bax and Bcl-2 [30,31]. Taken together, these results suggest that HRC-KD induces cardiac apoptosis through activation of CaMKII/p38 MAPK signaling pathways, causing deterioration of heart function under the heart failure conditions (Figure 9).

Discussion
According to the previous reports, HRC could interact with TRN and SERCA and regulate Ca 2+ cycling through inhibiting SERCA and RyR2 [4,8,9,10]. Chronic overexpression of HRC in mice resulted in increased TRN expression and inhibition of SERCA2 with hypertrophic phenotypes of increased fetal gene expression, HW/BW ratio, and fibrosis generation, indicative of a deterioration of cardiac function [11]. On the other hand, HRC overexpression could have also beneficial effects such as protection  of the heart from ischemia/reperfusion [32]. A previous HRC-KO study showed that ablation of HRC could not alter the Ca 2+ cycling properties, but on the other hand it could increase TRN protein expression [12]. The phenotypic changes at protein or functional levels occurring in response to overexpression or ablation of HRC could largely be associated with the adaptive or compensatory remodeling system-wide changes depending on the degree of expression of HRC [13]. The present study of HRC-KD consists of mainly two parts, 1) in vitro use of siRNA to knock down HRC in NRVCs and HL-1 cells to examine the direct effect of HRC on SERCA2 and RyR2 (Figures 1-4, and Supplemental Figures 1-3), and 2) in vivo use of adeno-associated virus (AAV) to knock-down HRC in the diseased heart to examine any beneficial effect. We generated an AAV-mediated partial HRC-KD system targeted to mouse heart to explore the physiological role of HRC in the heart to minimize the secondary effects of other proteins upor down-regulated during the remodeling processes of the gene targeted animals (Figure 1 and Figure S1). Our studies of HRC using the partial KD systems appears to be beneficial and provided several novel findings as follows: 1) Both RyR2 and SERCA activities were enhanced by KD of HRC (Figures 1, 4 and Figure  S3), 2) The increased RyR2 activity could directly cause the increased resting and peak Ca 2+ concentrations (Figures 2, 3 and  S2), 3)) Heart failure (HF) induced by TAC was further exacerbated by HRC-KD ( Figure 6) and 4) The exacerbated HF by HRC-KD is due at lease in part to the cytosolic Ca 2+ -leak and enhanced mitochondrial death pathways (Figures 7 and 8). Collectively, the present study suggests that the precise control of the expressional level of HRC is essential for keeping the normal functions of the heart.

TAC-associated Deterioration of Cardiac Function in HRC-KD Mice
Since HRC-KD showed increased Ca 2+ transient amplitude and enhanced activities of RyR2 and SERCA2, suggestive of an enhanced Ca 2+ cycling phenotype (Figures 2-4 and Figures S2-S3) in both neonatal rat ventricular cells (NRVCs) and HL-1 cells, we expected that HRC-KD would enhance cardiac function and may halt remodeling under pathological conditions such as the hypertensive hypertrophy model. However, HRC-KD alone did not rescue or decelerate cardiac function. On the contrary, it was associated with severe ventricular dilation and significantly decreased fractional shortening, indicative of further deterioration of cardiac function in the TAC model ( Figures 6A and 6B). The partial HRC-KD also resulted in cardiac fibrosis ( Figure 6C) and increased rates of cardiac cell death under the heart failure condition (Figure 7) potentially causing severe cardiac fibrosis [21]. The HRC expression was significantly reduced in the control (shNC) heart failure condition ( Figure 5D), similar to the previous finding [10]. The additional down-regulation of HRC mediated by AAV9-shHRC showed further deterioration of cardiac function, even though HRC-KD was partial, suggesting that a proper expression of HRC could be a pivotally important for keeping the heart in healthy conditions.

Activation of CaMKII-p38 MAPK Pathway and Increased Cardiac Cell Death in HRC-KD Hearts
Although the Ca 2+ cycling is enhanced by HRC-KD, the possible predominant effect of HRC on RyR2 as compared with the effect on SERCA could result in the increase in the cytosolic Ca 2+ concentration. Increases in cytosolic Ca 2+ concentration have been reported to induce activation of CaMKII, which is related to the transition from pressure overload-mediated hypertrophy to heart failure in mice [26,29]. In addition, the expression level and activity of CaMKII are highly up-regulated in heart failure [23,24,33,34]. In this study, we observed that the phosphorylation of CaMKII and p38 MAPK was highly increased in both heart failure groups, shNC failure and shHRC failure, mediated by sustained pressure overload; however, the phosphorylation of CaMKII was more extensive in the shHRC failure group than it was in the shNC failure group, suggesting the increased activity of CaMKII in HRC-KD hearts (Figure 8). We also observed that the phosphorylation of CaMKII was slightly, but significantly, increased in shHRC sham compared with shNC sham (160.05 in shNC sham vs. 1.2260.18 in shHRC sham) and there was a tendency to increase the phosphorylation level of PLB, RyR2 and p38 MAPK, however, it was not statistically significant. This might be caused by insufficient increase of CaMKII phosphorylation in shHRC sham heart. CaMKII is known to activate p38 MAPK [35], which induces mitochondrial death signaling by creating an imbalance between the expression of Bax and Bcl-2 (pro-and anti-apoptotic proteins, respectively) [30,31]. We also found that phosphorylation level of p38 MAPK was increased in the shHRC failure group compared with shNC failure group (Figure 8), which could induce an imbalance between Bax and Bcl-2 expression and lead to increased apoptosis in the hearts (Figure 7).

HRC as an Important Regulator for Cardiac Function
The previous HRC-KO mice showed impaired weight gain and TRN overexpression [12]. Furthermore, this animal model was susceptible to isoproterenol (ISO)-induced cardiac hypertrophy. These increased hypertrophic responses under conditions of cardiac stress are consistent with a regulatory role for HRC in SR Ca 2+ cycling in vivo. Thus, alterations in HRC levels, combined with additional genetic or environmental factors, may contribute to pathological hypertrophy and heart failure. Recently, a genetic variant of HRC-Ser96Ala-showed a significant correlation with ventricular tachycardia and sudden cardiac death in patients with idiopathic dilated cardiomyopathy [36,37]. Furthermore, reductions in HRC expression level may interrupt intracellular Ca 2+ homeostasis, leading to the development of heart failure [10]. On the other hand, overexpression of HRC could protect against ischemia/reperfusion induced cardiac injury [32]. Taken together, maintenance of HRC expression in the heart properly is important for maintaining the cardiac function, as HRC acts as an important regulator for cardiac performance. It will be interesting to see whether AAV-mediated HRC over-expression could restore cardiac function in the failed heart, since the expression level of HRC is substantially down-regulated in the TAC animals ( Figure 5).
In conclusion, HRC plays an important role in the regulation of Ca 2+ cycling, including SR Ca 2+ uptake and release, through the direct interaction with SERCA and TRN, respectively. It may also have a role in the progression of heart failure under pathophysiological conditions through stress-induced apoptotic pathways including CaMKII and p38 MAPK (Figure 9). Development of a method to maintain the proper expression level of HRC will likely be crucial for management of the heart diseases in the future.

Ethics Statement
All animal experiments were approved by the Gwangju Institute of Science and Technology Animal Care and Use Committee (the permit number: GIST-2011-1).

HL-1 Cell Culture
HL-1 cells obtained as a kind gift from Dr. W. Claycomb (Louisiana State University Medical Center) were maintained as described previously [38]. Briefly, cells were cultured on gelatin (0.02%, w/v)/fibronectin (10 mg/ml)-coated cell culture plates. The cells were maintained in Claycomb medium (SAFC BIOSCIENCES TM ) supplemented with 10% fetal bovine serum (Sigma-Aldrich Co.), 2 mM L-glutamine, 0.1 mM norepinephrine, 100 unit/ml penicillin, and 100 mg/ml streptomycin (Invitrogen). The culture medium was changed with fresh medium every 24 hours. The cells were grown at 37uC in an atmosphere of 5% CO 2 and 95% air in an incubator.

Primary Cell Culture and siRNA oligo Transfection
Primary cultures of neonatal rat ventricular cells (NRVCs) from 2-day-old Sprague-Dawley rats were prepared as described [39]. For siRNA study, the oligonucleotides and transfection reagents were purchased from Thermo Fisher Scientific, Inc. The predesigned ON-TARGET plus SMART pool was used for knock-down of rat HRC gene. siRNA transfection was performed as described previously [40].

Calcium Transient Measurement
Ca 2+ transients in NRVCs or HL-1 cells were measured as described previously [40]. Briefly, siRNA transfected NRVCs or HL-1 cells on glass coverslips were incubated with Fura-2 AM (Molecular Probes) in Tyrode solution containing 10 mM HEPES-NaOH, pH 7.4, 135 mM NaCl, 4.0 mM KCl, 1.0 mM MgCl 2 , 1.8 mM CaCl 2 , and 10 mM glucose for 30 min and washed in dye-free Tyrode solution. The cells were placed in a circulating bath with Tyrode solution held at 37uC under an inverted microscope. A dual-beam excitation spectrofluorometer setup (IONOPTIX) was used to record fluorescence emissions (505 nm) elicited from exciting wavelengths of 340 and 380 nm. Ca 2+ transient amplitude measured as fluorescence ratio (340:380 nm), cytosolic free Ca 2+ concentration (baseline), time required to reach 50% of baseline (T 50 ), and time to peak of Ca 2+ transients were acquired. SR Ca 2+ content was estimated by rapid application of 20 mM caffeine in Ca 2+ -free Tyrode solution. Data were analyzed by using Ion Wizard software (IONOPTIX). In the experiment shown in Figure 3, 1 mM isoproterenol (ISO) was used.

Microsome (SR) Preparations from HL-1 Cells
Microsome preparations were carried out as described previously [41] with some modification. HL-1 cells grown on cell culture plate were washed with 5 ml of D-PBS (pH 7.4, WelGENE Inc., South Korea) containing protease inhibitor cocktail (Roche Applied Science) and harvested in the same solution by scraping. Cells were collected by centrifugation at 5500 rpm for 10 min in a Sorvall SS-34 rotor. Cell pellets were lysed with lysis buffer containing 0.6% phosphatydylcholine, 1 M Tris-HCl, pH 7.4, 0.1 M HEPES, 1 M NaCl, 1% CHAPS and 1X protease inhibitor cocktail, and incubated using rotatory incubator on 4uC. Lysed HL-1 cells were centrifuged at 5500 rpm for 10 min and the supernatant was centrifuged at 43000 rpm in a Beckman Ti-70 rotor for 60 minutes. The pellets were homogenized with storage buffer containing 1 M sucrose, 0.1 M HEPES, pH 7.4, 1 M KCl and 1X protease inhibitor cocktail in a Teflon-glass Dounce homogenizer and stored at 280uC. Protein concentration of the microsome was measured using BCA Protein Assay kit (Thermo Scientific). Tritium Ryanodine Binding Assay [ 3 H]ryanodine binding assay in NRVCs or HL-1 cells was performed as described previously [42,43] with some modifications. Briefly, equilibrium ryanodine binding to whole homogenate Oxalate-supported Ca 2+ Uptake Assay Ca 2+ uptake rate was measured in siRNA transfected NRVCs or HL-1 cells as described previously [44]. Briefly, NRVCs or HL-1 cells were resuspended in the homogenization buffer containing 50 mM KH 2 PO 4 , 10 mM NaF, 1 mM EDTA, 0.3 M sucrose, 1X proteinase inhibitor cocktail and 0.5 mM DTT, and homogenized by Dounce glass homogenizer. 250 mg of cell lysates was added to 2.2 ml of the uptake buffer containing 100 mM KCl, 5 mM MgCl 2 , 5 mM NaN 3 , 0.5 mM EGTA, 1 mM ruthenium red, 200 mM CaCl 2 and 40 mM imidazole, pH 7.0, and the reaction mixtures were incubated at 37uC for 4 min. The uptake reactions were initiated by serial addition of 5 mM K-oxalate and 5 mM Mg-ATP. Aliquots were filtered through a 0.45 mm Millipore filter after 1, 2, 3 and 4 minutes to terminate the reaction. The initial rate of Ca 2+ uptake was calculated by linear regression analysis of the uptake values at 1, 2, 3 and 4 minutes. The results were analyzed using SigmaPlot 10 software. Figure 9. A flow-chart showing the putative mechanism for the deteriorated cardiac function by HRC-KD in TAC-HF model. Downregulation of HRC expression induces increased RyR2 and SERCA2 activities followed by an increase of cytosolic Ca 2+ , which leads to activation of CaMKII and increased mitochondrial Ca 2+ uptake. The activated CaMKII phosphorylates downstream kinases and causes cardiac cell death through an imbalance of the Bax/Bcl-2 ratio. doi:10.1371/journal.pone.0043282.g009

Transverse Aortic Banding
All animal protocols were approved by GIST Animal Care and Use Committee (the permit number: GIST-2011-1) and conform to the Guideline for the Care and Use of Laboratory Animals published by the United States National Institutes of Health. Male mice (C57BL/6) of 9 weeks old (23-25 g) were used for the present study. The animals were anesthetized with 0.3-0.5 ml of 1X Avertin solution (mixture of 2-2-2 tribromoethanol and tert-amyl alcohol) by intraperitoneal injection. Mice were ventilated with a tidal volume of 0.1 ml and a respiratory rate of 120 breaths per minute (Harvard Apparatus). A 2-to 3-mm longitudinal cut was made in the proximal portion of the sternum which allowed visualization of the aortic arch. The transverse aortic arch was ligated between the brachiocephalic and left common carotid arteries with an overlaying 27-gauge needle, and then the needle was immediately removed leaving a discrete region of constriction. One week post-operation mortality was less than 10%.

Histological Analysis of Hearts
Mice were euthanized by cervical dislocation under 2-2-2 tribromoethanol/tert-amyl alcohol anesthesia. Excised hearts were fixed in 4% formalin for 72 hours, embedded in paraffin, and sectioned (10 mm). Trichrome staining of the sectioned hearts was performed to measure the fibrotic areas. The fibrotic areas stained blue and the normal tissue stained red. Apoptosis was examined using the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) assay kit (In Situ Cell Death Detection Kit, TMR red; Roche Applied Science) according to manufacturer's instructions. TUNEL-positive nuclei in the heart section were calculated at X40 magnification under LSM-700 confocal microscope (Carl Zeiss Co., Ltd.).