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SPEG (Striated Muscle Preferentially Expressed Protein Kinase) Is Essential for Cardiac Function by Regulating Junctional Membrane Complex Activity

Originally published Research. 2017;120:110–119



Junctional membrane complexes (JMCs) in myocytes are critical microdomains, in which excitation–contraction coupling occurs. Structural and functional disruption of JMCs underlies contractile dysfunction in failing hearts. However, the role of newly identified JMC protein SPEG (striated muscle preferentially expressed protein kinase) remains unclear.


To determine the role of SPEG in healthy and failing adult hearts.

Methods and Results:

Proteomic analysis of immunoprecipitated JMC proteins ryanodine receptor type 2 and junctophilin-2 (JPH2) followed by mass spectrometry identified the serine–threonine kinase SPEG as the only novel binding partner for both proteins. Real-time polymerase chain reaction revealed the downregulation of SPEG mRNA levels in failing human hearts. A novel cardiac myocyte-specific Speg conditional knockout (MCM-Spegfl/fl) model revealed that adult-onset SPEG deficiency results in heart failure (HF). Calcium (Ca2+) and transverse-tubule imaging of ventricular myocytes from MCM-Spegfl/fl mice post HF revealed both increased sarcoplasmic reticulum Ca2+ spark frequency and disrupted JMC integrity. Additional studies revealed that transverse-tubule disruption precedes the development of HF development in MCM-Spegfl/fl mice. Although total JPH2 levels were unaltered, JPH2 phosphorylation levels were found to be reduced in MCM-Spegfl/fl mice, suggesting that loss of SPEG phosphorylation of JPH2 led to transverse-tubule disruption, a precursor of HF development in SPEG-deficient mice.


The novel JMC protein SPEG is downregulated in human failing hearts. Acute loss of SPEG in mouse hearts causes JPH2 dephosphorylation and transverse-tubule loss associated with downstream Ca2+ mishandling leading to HF. Our study suggests that SPEG could be a novel target for the treatment of HF.


Heart failure (HF) is a deadly and costly disease affecting 5.7 million people in the United States alone.1 Systolic cardiac contraction and diastolic relaxation depend on proper calcium (Ca2+) signaling because cardiac myocytes translate electric signals to mechanical contraction via Ca2+ signaling, a process known as excitation–contraction coupling (ECC). ECC occurs within the specialized subcellular structures called junctional membrane complexes (JMCs), in which voltage-gated L-type Ca2+ channels on the invaginations of the plasmalemma known as transverse tubules (TTs) are positioned near the major cardiac Ca2+ release channels ryanodine receptors (RyR2) on the sarcoplasmic reticulum (SR).2 Disruptions in the JMC can lead to hyperactivation of RyR2s causing abnormal Ca2+ release from the SR, which has been implicated in the development of HF.3 We recently demonstrated that junctophilin-2 (JPH2) is a protein essential for the formation and maintenance of JMC structures and TTs within cardiac myocytes.4,5

Editorial, see p 11

In failing hearts, reduced JPH2 protein levels have been associated with a loss of JMCs because of the loss of TT number and regularity, resulting in impaired ECC.4 Reduced JPH2 levels can also enhance RyR2 channel activity because JPH2 binding to RyR2 stabilizes its closed state.6 Numerous studies have shown that altered post-translational modulation of RyR2 also plays an important role in HF, including enhanced phosphorylation and oxidation of the pore-forming channel subunits.7,8 This increase in RyR2 activity has been associated with diastolic SR Ca2+ leak, which may contribute to SR Ca2+ depletion and impaired contractility. However, the mechanisms underlying altered RyR2 regulation and JMC deterioration in HF remain incompletely understood.

Here, we used unbiased proteomic analysis of immunoprecipitated RyR2 and JPH2 from adult mouse hearts to identify novel proteins within the JMC. A protein known as SPEG (striated muscle preferentially expressed protein kinase)9 was identified as the only protein that binds to both RyR2 and JPH2 in the mouse heart. SPEG—a myosin light chain kinase family protein—contains dual serine/threonine kinase domains unique to the obscurin subfamily, as well as multiple immunoglobulin-like, fibronectin type III domains, and proline-rich regions typically involved in protein–protein interactions.9SPEG is a single gene, which is alternatively spliced into several tissue-specific isoforms, including APEG (aortic preferentially expressed gene; aorta), BPEG (brain preferentially expressed gene; brain), and SPEGα and SPEGβ (skeletal and cardiac muscle). It has been previously demonstrated that SPEG is important for cardiac development because a germline knockout of all SPEG isoforms resulted in embryonic dilated cardiomyopathy and early postnatal death.10 In addition, rare SPEG mutations have been implicated in centronuclear myopathy, a genetic disease of skeletal and cardiac muscle.11 Importantly, however, nothing is known to date about the potential role of SPEG in the adult heart or the mechanisms by which altered SPEG levels modulate cardiac dysfunction.

On the basis of SPEG’s interaction with key proteins within the JMC and its importance in cardiac development, we hypothesized that cardiac isoforms of SPEG are necessary for normal adult cardiac function and ECC. Therefore, we tested the role of SPEG in adult hearts and nongenetic forms of HF using a novel inducible cardiac myocyte-restricted Speg knockout mouse. Our data revealed reduced SPEG levels in patients with HF and development of HF in inducible cardiac-specific Speg knockout mice. These data demonstrate for the first time that SPEG plays a critical role in the maintenance of JMC integrity and SR Ca2+ handling in adult mammalian hearts and suggest that loss of SPEG contributes to the development of excitation–contraction deficits in HF. In addition, our data revealed that loss of SPEG causes disruption of the TT network before HF development, despite unaltered JPH2 levels. Loss of SPEG-mediated JPH2 phosphorylation was found to be associated with TT disarray, which suggests that defective post-translational modulation of JPH2 by SPEG represents a key early event in the pathogenesis of HF.


Study Animals

All mouse studies were performed according to the protocols approved by the Baylor College of Medicine Institutional Animal Care and Use Committee and conform to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. For the proteomics experiments, whole ventricles were harvested from wild-type (WT) C57Bl/6J mice and hemagglutinin (HA)-tagged JPH2 overexpressing mice6 between the ages of 3 and 6 months and immediately flash-frozen in liquid nitrogen. Speg chimeric mice were obtained from the knockout mouse project at University of California (Davis, CA). These mice were crossed to Flippase mice (The Jackson Laboratory, Bar Harbor, ME) to develop a conditional Speg allele. Conditional Speg mice were crossed to Myh6-driven mER-Cre-mER (MCM) mice (kindly provided by Dr Jeffrey Molkentin) to create cardiac myocyte-specific tamoxifen-inducible Speg knockout mice. Male MCM-Spegfl/fl and MCM controls injected with tamoxifen for 5 days were used for the studies described in our article.

Mass Spectrometry

Coimmunoprecipitation followed by mass spectrometry (IP-MS) experiments were performed as previously described with few modifications.12 Briefly, RyR2 was immunoprecipitated from WT mouse hearts using RyR2 monoclonal antibody (MA3-916; Thermo Fisher Scientific, Waltham, MA), and JPH2 was immunoprecipitated from JPH2 overexpressing hearts using monoclonal HA antibody (Sigma-Aldrich, St. Louis, MO). Murine nonspecific IgG (Sigma-Aldrich) and beads were used as negative controls. Immunoprecipitates were run on a gel, and each lane was cut and trypsinized followed by tandem MS/MS and analysis with Maxquant software. To distinguish between specific and nonspecific binding, we used a cutoff ratio of 3 for the intensity of each protein that came down with the RyR2 IP versus IgG IP and RyR2 IP versus beads only IP and a ratio of 4.5 for the same with JPH2 IP versus IgG IP and JPH2 IP versus beads only IP. A table of putative binding partners (Online Tables I and II) was generated with the most specific binding partners highlighted in grey (red dots in Figure 1A and 1B).

Figure 1.

Figure 1. SPEG (striated muscle preferentially expressed protein kinase) directly binds to ryanodine receptor type 2 (RyR2) and junctophilin-2 (JPH2) within junctional membrane complexes. A and B, Unbiased proteomics analyses of putative RyR2 and JPH2 binding partners based on adult mouse heart coimmunoprecipitation of RyR2 and JPH2, respectively, coupled to mass spectrometry (IP-MS). Specific binding partners (red dots) were defined based on cutoff ratios of 3 and 4, respectively, of the MS intensity of each protein that came down with RyR2 or JPH2 IP vs control IgG IP (y axis) and RyR2 or JPH2 IP vs beads only IP (x axis). C, Schematic overview of SPEG protein structure with key functional domains marked and SPEG fragments used for binding site analysis. D, Western blots of reciprocal SPEG co-IP experiment from mouse heart showing that both RyR2 and JPH2 but not SERCA2a (sarcoplasmic endoplasmic reticulum ATPase type 2a) pulled down with SPEG. E, RyR2, Myc-tagged SPEGβ N-terminal peptide, and FLAG-tagged SPEGα were coexpressed in HEK293 cells. Only SPEGβ N-terminal peptide was shown to bind the RyR2 pull-down. F, JPH2 was coexpressed with Myc-tagged SPEGβ N-terminal peptide and FLAG-tagged SPEGα; only FLAG-tagged SPEGα was shown to bind the JPH2 pull-down.


cDNA encoding SPEGα and the N-terminal 861 amino acids of SPEGβ was reverse transcribed from RNA isolated from WT mouse ventricle. Speg inserts were amplified using polymerase chain reaction with KOD Xtreme Hot Start Kit (EMD Millipore) and the following primers: Spegα+N-terminal FLAG-tag: Fwd-5′CTGAATGATATCGCCACCATGGATTACAAGGATG-ACGATGACAAGGACGTCAAGCTCAGTCCCAGCCAGGATCATGATTCC-3′; Rev-5′-CTGAATGCGG-CCGCCTAGGGGCTGCCAGGGTAGGAGCGG-3′. Spegα was cloned into a pcDNA3.1+ vector using restriction sites EcoRV and NotI. HA-tagged Jph2 was cloned from mouse heart using the following primers: Fwd-5′-AAGAATTCGCCACCATGTACCCATACGACGTCCCAGACTACGCTAGCGGGGGCC-GCTTTGACTTTG-3′ and Rev-5′-AATCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTA AGTCAGGAGGTGAACAAATAGG-3′ using EcoRI and XbaI restriction digest. Constructs were cloned into a pcDNA3.1+ vector using T4 ligase (NEB). The Spegβ N terminus was amplified using the following primers: Fwd-5′-ATCTAAGCTTCAGAAAGCCCGGGGCACG-3′ and Rev-5′-ATTAGCGGCCGCCT-ACTGGCTGGGACTGAGCTTC-3′ and was cloned into a cMyc-tagged pcDNA3.1+ vector using the HindIII/ NotI restriction digest (NEB) followed by ligation with T4 ligase for 10 minutes at room temperature. Constructs were transformed into MAX Efficiency DH5α (Thermo Fisher Scientific) competent cells. Recombinant pCMV5-hRyR2 was kindly provided by Dr Andrew Marks.

Cell Culture and Recombinant DNA Expression

HEK293T cells were seeded into 10 cm plates and grown in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum, 1% penicillin–streptomycin, and 1% L-glutamine. Plasmids encoding full-length RyR2, HA-tagged JPH2, cMyc-tagged SPEGβ N terminus (amino acids 1–861), or full-length FLAG-tagged SPEGα were coexpressed in HEK293T cells. Cells were washed in ice cold phosphate-buffered saline (PBS) and harvested by scraping 60 hours post transfection. Cells were spun down and resuspended in a modified radioimmunoprecipitation assay (RIPA) buffer containing 1% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), PhosSTOP phosphatase inhibitor, and cOmplete mini protease inhibitor cocktail (Roche), 20 mmol/L NaF and 1 mmol/L Na3VO4. Samples were sonicated 3× for 1 s each on ice and centrifuged at 16 000 g for 15 minutes. Supernatants were collected as the cell lysate.

Co-IP and Western Blots

Reciprocal SPEG co-IP from mouse heart lysate was performed using the same protocol as the co-IP for mass spec, except that SPEG polyclonal, rabbit antibody (custom-made by Yenzyme), was used to immunoprecipitate SPEG, and rabbit IgG was used as a negative control. After washing 2× with modified RIPA buffer, beads were incubated with 2× Lamelli buffer at room temp for 30 minutes then gel electrophoresed, transferred to a polyvinylidene fluoride (PVDF) membrane, and immunoblotted. For JPH2 phosphoserine Western blots (WBs), Dynabeads (Invitrogen) and JPH2 custom antibody (Yenzyme) were used to coimmunoprecipitate JPH2 from MCM and MCM-Spegfl/fl heart lysates before WB and probing with phosphoserine followed by total JPH2 antibody.

For co-IP from HEK cells, 500 μg of cell lysate was rotated overnight at 4°C in 500 μL of RIPA) buffer with 2 μL of either monoclonal anti-RyR2 (ma3-916) to pull down RyR2, anti-FLAG (M2, Sigma-Aldrich), or anti-HA (Sigma-Aldrich) to pull down JPH2.6 Murine IgG nonspecific antibody was used as a negative control. Next, 40 μL of PBS-washed protein G beads (Pierce) resuspended in RIPA was incubated with lysate–antibody complexes for 1.5 hours at 4°C. Bead–antibody–protein complexes were washed 2× using the same modified RIPA buffer containing 0.5% CHAPS. Beads were incubated in 2× Lamelli buffer for 10 minutes at 70°C. The immunoprecipitates and 75 μg of input lysate were gel electrophoresed and immunoblotted with the appropriate antibodies to confirm pull-down and either anti-cMyc (9E10, Sigma-Aldrich), anti-FLAG, anti-RyR2, or anti-JPH2 to determine which SPEG isoform coimmunoprecipitated with RyR2 and JPH2.

For WB from mouse heart, mouse heart lysate was made by first flash freezing hearts in liquid nitrogen and homogenizing them in the modified RIPA buffer listed above before sonication for 3× for 1 s. After gel electrophoresis on a 6% to 12% acrylamide gel, proteins were transferred to a PVDF membrane overnight. Membranes were blocked 30 minutes with 3% bovine serum albumin (BSA)-tris–buffered saline (TBS) and incubated overnight at 4°C in custom antibodies (Yenzyme) for SPEG, p-S2808 and p-S2814, or antiphosphoserine (Sigma) diluted 1:1000 in 3% BSA-TBS. JPH2 (Yenzyme) and SERCA2a (sarcoplasmic endoplasmic reticulum ATPase; ma3-919, Thermo Fisher) were diluted in 5% milk–TBS and applied to SPEG IP WBs. Blots were then incubated in the appropriate fluorescent secondary antibodies (anti-rabbit, Rockland; anti-mouse, Thermo Fisher Scientific) diluted 1:10 000 in 5% milk–TBS and visualized. Total RyR2 (MA3-916) or total JPH2 (custom, Yenzyme) were subsequently assessed.

Human Heart Samples

Left ventricular samples were obtained from patients undergoing clinically indicated cardiac transplantation for end-stage HF after obtaining informed consent (Online Table III). Seven nonfailing human hearts (which could not be used for transplantation) were obtained from the International Institute for the Advancement of Medicine. All studies were performed with institutional review board approval from Baylor College of Medicine and Texas Heart Institute (H31918).

Quantitative Real-Time Polymerase Chain Reaction

RNA isolation and quantitative real-time polymerase chain reaction were performed as previously described.13 RNA was isolated from human left ventricle samples using the Zymo Directzol miniprep kit. cDNA was prepared from the resulting RNA using iScript. quantitative real-time polymerase chain reaction was performed using the following human SPEG primers: exons 9 to 10 Fwd-5′-GCGGTCAATGAGTATGGTGC-3′ and Rev-5′-CATTTGAGCAGTGCAGGCTG-3′. Samples were assayed in triplicate and normalized to L7.


Mouse cardiac samples were fixed in 10% buffered formalin and dehydrated in an ethanol series. Histoclear and paraffin washes were used to paraffinize the samples. Five micrometer sections were cut, placed on slides, and deparaffinized. Sections were stained with Masson trichrome (Thermo Fisher Scientific) and imaged on a light microscope to highlight gross morphology and fibrosis.


Echocardiography was performed using the Vevo 770 and 2100 imaging systems (VisualSonics, Toronto, Canada) equipped with a high-frequency probe.14 Measurements were made while the mouse was anesthetized using 2± 0.5% isoflurane and had a heart rate >400 bpm. In addition, body temperature was maintained in a range (37.0±1.0°C) to avoid confounding effects of hypothermia.

Calcium Imaging Studies

Mice from each group were anesthetized with 2% isoflurane and humanely euthanized by cervical dislocation. Cells were isolated from hearts by enzymatic digestion with liberase using Langendorff perfusion and mechanical shearing of the ventricles. Ventricular myocytes were then incubated in either di-8-ANNEPS (amino-naphthyl-ethylene-pyridinium-propyl-sulfonate) or Fluo-4 am and J127 and subsequently imaged by confocal microscopy.5 Line scans were used to obtain Ca spark data and transient amplitudes. Caffeine was used to determine the SR load. Data were analyzed using Image J and the SparkMaster plug-in.

TT Imaging and Analysis

TTs were visualized in ventricular myocytes using di-8-ANEPPS staining (10 μmol/L for 10 minutes) in normal Tyrode solution containing 1.8 mmol/L Ca2+. The spatial integrity of TTs was quantified using methods described in previous studies4 and was performed with ImageJ software ( The regions of interest were selected within a cell and excluded the nucleus. The power spectrum was computed using fast Fourier transform. The normalized power at a spatial frequency of ≈0.55 μm1 (peak power at ≈0.55 μm1 normalized to average power at a spatial frequency of 0.2–0.4 μm1) was used as an index of the spatial integrity of TTs (TT power).

Immunocytochemistry in Isolated Myocytes

Cardiac myocytes were isolated from C57Bl/6 WT mice as described for the Ca2+ imaging studies. Cells were then attached to a slide with laminin and fixed using 4% paraformaldehyde. Tritin-X was used to permeabilize the cells, which were blocked with normal goat serum (NGS) before incubating in primary antibody. Primary antibodies against SPEG (Yenzyme) and RyR2 (MA3-916) diluted in 1%NGS/1%BSA were incubated with cells overnight at 4°C. Cells were washed in PBS and incubated with Alexa 488 goat-anti-mouse and 568 goat-anti-rabbit secondary antibodies. Cells were imaged by confocal microscopy.

Statistical Analysis

Statistical differences in the survival of MCM and MCM-Spegfl/fl mice were assessed using the log-rank test. For all other statistics, values were expressed as mean±SEM. Statistical differences between MCM and MCM-Spegfl/fl mice were tested for by unpaired t test for a particular time point or multiple t tests between time points. A value of P <0.05 was considered statistically significant.


SPEG Is a Novel JMC Protein Associated With RyR2 and JPH2

To identify novel regulatory proteins within the JMC, we performed unbiased proteomic analyses of proteins that bind to RyR2 or JPH2, respectively. RyR2 and JPH2 were immunoprecipitated separately from mouse ventricular lysates. Next, binding partners of RyR2 and JPH2 were identified using MS. About 20 proteins were found to be associated with RyR2 or JPH2 in adult mouse hearts (Figure 1A and 1B). Because conditions of the IP were stringent, we did not recover many of the known members of the RyR2 macromolecular complex, presumably because their weak interactions. Instead, several new binding partners were recovered with higher confidence (Online Tables I and II). The most relevant finding for this investigation, however, was the identification of SPEG as the only protein that bound specifically to both RyR2 and JPH2.

To validate that SPEG directly binds to RyR2 and JPH2, we performed the reciprocal co-IP experiment in WT mouse heart lysate. SPEG was immunoprecipitated from C57Bl/6N mouse hearts lysate, and immunoblot confirmed that SPEG binds both RyR2 and JPH2 but not another SR membrane protein sarcoplasmic endoplasmic reticulum ATPase type 2a (SERCA2a; Figure 1D). In addition, we demonstrated that SPEG colocalizes with RyR2 within isolated cardiac myocytes (Online Figure I). To further examine RyR2 and JPH2 binding to SPEG, we performed coexpression and co-IP studies in HEK293 cells of full-length RyR2 or JPH2 with SPEG fragments. Myc-tagged SPEGβ N-terminal and FLAG-tagged SPEGα fragments were coexpressed with full-length RyR2 or HA-tagged JPH2. RyR2 was pulled down from HEK293 lysates with nonspecific mouse IgG as negative control. WBs demonstrated RyR2 interaction with the N-terminal SPEGβ fragment (Figure 1E). In contrast, JPH2 pull-down with anti-HA antibody revealed an interaction with SPEGα (Figure 1F). These experiments demonstrate that SPEG binds RyR2 and JPH2 independently and via distinct domains, which corresponds to the SPEG isoforms identified in the IP-MS experiment.

Reduced SPEG Levels in Patients With HF

Recent studies linked rare loss-of-function mutations in SPEG to dilated cardiomyopathy in humans.11 We measured SPEG mRNA levels in patients with nongenetic forms of HF (Online Table III). Quantitative polymerase chain reaction revealed an 83% reduction in SPEG transcripts compared with nonfailing human hearts (Figure 2A). To circumvent prenatal lethality in germline Speg knockout mice10 and study the role of SPEG in cardiac myocytes, we obtained generated conditional cardiac myocyte-restricted tamoxifen-inducible Speg knockout (MCM-Spegfl/fl) mice by crossing Speg-floxed (fl) mice with Myh6-driven MCM mice. On administration of tamoxifen for 5 days, SPEG protein levels were assessed using Western blotting in the hearts of MCM-Spegfl/fl and MCM control mice (Figure 2B). Quantification of the protein levels at 2 weeks after tamoxifen revealed a robust 80% reduction in cardiac isoforms of SPEG in the hearts of MCM-Spegfl/fl mice compared with controls (Figure 2C).

Figure 2.

Figure 2. SPEG (striated muscle preferentially expressed gene) is downregulated in human heart failure (HF) and a mouse model of cardiac myocyte specific inducible SPEG knockout. A, Quantitative real-time polymerase chain reaction analysis of SPEG mRNA transcript abundance in myocardium from patients with HF or patients with normal heart function (NF). B, SPEG Western blots in hearts from inducible cardiac-specific SPEG-deficient mice (MCM-Spegfl/fl) and MCM control mice, and C, quantification of SPEG levels in MCM and MCM-Spegfl/fl knockout mice. *P<0.05, ***P<0.001.

Inducible Cardiac Myocyte-Specific SPEG Deficiency Causes HF

To assess the effects of cardiac myocyte-specific SPEG deficiency on heart function, we prospectively followed cohorts of MCM-Spegfl/fl (n=9) and MCM control mice (n=10) after tamoxifen injections. Kaplan–Meier survival curve analysis revealed that the loss of SPEG leads to premature death in the MCM-Spegfl/fl (44% death by 8 weeks after tamoxifen induction; Figure 3A). Representative images of M-mode echocardiography performed at 8 weeks after tamoxifen showed evidence of weakened contractility and cardiac dilation in MCM-Spegfl/fl (Figure 3B and 3C). Quantification of serial echocardiography measurements before, 4, and 8 weeks after tamoxifen revealed that at baseline (before tamoxifen), there were no differences between both groups of mice (Figure 3D and 3E). Starting at 4 weeks and progressively deteriorating at 8 weeks post tamoxifen, SPEG deficiency led to the development of contractile dysfunction as evidenced by a decline in the ejection fraction (Figure 3D). In addition, there was evidence of left ventricular dilatation at 4 weeks, which became much more pronounced at 8 weeks post tamoxifen (Figure 3E). Online Table IV demonstrates that cardiac dilatation of the anterior and posterior wall, as well as the intraventricular septum, occurred by 8 weeks post tamoxifen. Final, histological analysis of Masson trichrome-stained sections of hearts obtained at 8 weeks revealed evidence of severe dilated cardiomyopathy with thinning of the ventricular walls and pronounced left atrial fibrosis (Figure 3F). Together, these findings demonstrate that conditional loss of SPEG is causally linked to HF development in MCM-Spegfl/fl mice.

Figure 3.

Figure 3. SPEG (striated muscle preferentially expressed protein kinase) downregulation causes heart failure. A, Kaplan–Meier survival plot showing percent survival of MCM and MCM-Spegfl/fl mice for 8 weeks after tamoxifen injection. B and C, Representative M-mode echocardiography images from MCM and MCM-Spegfl/fl knockout mice at 8 weeks post tamoxifen. Summary bar graphs showing (D) left ventricular ejection fraction (EF) and (E) left ventricular internal diameter in systole (LVID; s) at baseline (pre) and 4 or 8 weeks after tamoxifen injection. F (i and ii), Representative images of whole hearts from MCM (i) and MCM-Spegfl/fl (ii) mice after tamoxifen and (iii and iv) Masson trichrome histological images of MCM (iii) and MCM-Spegfl/fl (iv) hearts. G and H, Quantification of heart weight (HW; G) and lung weight (LW; H) to body weight (BW). *P<0.05, **P<0.01, ***P<0.001 vs MCM control.

Aberrant Systolic SR Ca2+ Handling in SPEG-Deficient Mice

Because we found that SPEG binds to RyR2, we determined functional effects of SPEG deficiency on SR Ca2+ handling and RyR2 activity in situ. Ventricular myocytes were isolated from MCM-Spegfl/fl mice 8 weeks after tamoxifen injection and paced at 1 Hz to obtain steady-state Ca2+ handling. There was a reduction in steady-state Ca2+ transient amplitude in SPEG-deficient myocytes consistent with systolic dysfunction in Speg conditional knockout mice (Figure 4A and 4C). Rapid application of caffeine revealed a modest but significant reduction in SR Ca2+ load (Figure 4D). Quantification of the Ca2+ transient decline indicated that the reduced SR load was not because of a reduced SERCA2a activity (Figure 4E). Overall, these findings indicate that loss of SPEG negatively impacts SR Ca2+ load and systolic SR Ca2+ release, consistent with reduced contractility in SPEG-deficient mice.

Figure 4.

Figure 4. Abnormal systolic sarcoplasmic reticulum (SR) Ca2+ release in SPEG (striated muscle preferentially expressed protein kinase)–deficient cardiac myocytes. A and B, Representative tracings of Ca2+ transient recordings after 1 Hz pacing, subjected to caffeine at the end, in myocytes from MCM and MCM-Spegfl/fl knockout mice 8 weeks post tamoxifen. C, Quantifications of Ca2+ transient amplitude, (D) SR Ca2+ load, and (E) SERCA2a (sarcoplasmic endoplasmic reticulum ATPase type 2a) activity. *P<0.05.

Loss of SPEG Leads to Diastolic Ca2+ Dysregulation

To investigate whether diastolic Ca2+ handling was also affected in SPEG-deficient mice, we measured spontaneous SR Ca2+ release events in isolated cardiac myocytes using confocal microscopy (Figure 5A and 5B). Quantification of the data revealed an elevated Ca2+ spark frequency in SPEG-deficient myocytes, consistent with enhanced RyR2-mediated SR Ca2+ leak (Figure 5C). Further analysis of the Ca2+ spark properties revealed a slight increase in Ca2+ spark amplitude (Figure 5D) and a reduction in the full-width half-maximum (spatial width; Figure 5E). These data suggest that loss of SPEG directly alters RyR2 activity, which is associated with HF development. Although RyR2 hyperphosphorylation at S2808 and S2814 has been shown to contribute to Ca2+ dysregulation in HF progression,1416 the phosphorylation states of these sites were unaltered in MCM-Spegfl/fl hearts (Online Figure II). Thus, enhanced SR Ca2+ leak through hyperactive RyR2 may contribute to a reduced SR Ca2+ load and contractile dysfunction in SPEG-deficient hearts.

Figure 5.

Figure 5. Spontaneous ryanodine receptor type 2–mediated Ca2+ release in cardiac myocytes with loss of SPEG (striated muscle preferentially expressed protein kinase). A and B, Confocal microscopy line scans revealing increased number of Ca2+ sparks in MCM-Spegfl/fl mice 8 weeks post tamoxifen. C, Bar graphs showing quantification of Ca2+ spark frequency (CaSpF), (D) Ca2+ spark amplitude, and (E) full-width of half max (FWHM). *P<0.05, **P<0.01, ***P<0.001.

SPEG Is Essential for JMC Integrity

Our data also revealed that SPEG directly binds to JPH2, a protein critical for JMC stability in cardiac myocytes.4 To assess potential changes in JMC organization in SPEG-deficient ventricular myocytes, we stained the structure of the TT network using di-8-ANEPPS. A significant disruption of the normal TT structure was apparent in tamoxifen-treated MCM-Spegfl/fl mice after the onset of HF (Figure 6A through 6D). Image quantification revealed that SPEG deficiency causes cellular hypertrophy (Figure 6E) and a pronounced loss of TT power and TT area (Figure 6F and 6G) consistent with an overall loss of JMC integrity. Together, these data indicate that HF in MCM-Spegfl/fl mice is caused by knockdown of SPEG, and that SPEG is required for JMC integrity and normal ECC in cardiac myocytes.

Figure 6.

Figure 6. SPEG (striated muscle preferentially expressed protein kinase) knockdown causes disrupted junctional membrane complexes. A and B, Ventricular myocytes isolated from MCM-Spegfl/fl mice or MCM controls 8 weeks post tamoxifen stained with di-8-ANEPPS to visualize transverse-tubule (TT) organization. C and D, Enlarged image (corresponding to white box in A and B) showing disrupted TT structures in MCM-Spegfl/fl knockout mice. E, Quantification of ventricular myocyte size, (F) normalized TT power, and (G) TT area. *P<0.05, **P<0.01, ***P<0.001.

Loss of SPEG Phosphorylation of JPH2 Causes TT Disruption Preceding HF Development

To determine whether the aberrant Ca2+ handling and TT loss associated with SPEG deficiency are causally linked to HF development or merely a secondary consequence of HF, we studied another group of tamoxifen-treated MCM-Spegfl/fl mice at 2 weeks post tamoxifen. At this earlier time point, the average ejection fraction of MCM-Spegfl/fl mice was 47.4±3.3% compared with 55.7±1.7% in MCM controls (P<0.05; Online Figure III). Despite this small but significant decline in cardiac contractility, the MCM-Spegfl/fl mice were not in HF. Consistent with these findings, there was a modest reduction in the Ca2+ transient amplitude in ventricular myocytes from MCM-Spegfl/fl mice compared with MCM controls (Online Figure IVA through IVC). However, there were no significant changes in SR Ca2+ load (Online Figure IVD), SERCA2a activity (Online Figure IVE), or SR Ca2+ spark frequency (Online Figure IVF through IVH).

In contrast, di-8-ANEPPS staining revealed severely disrupted TT structures in ventricular myocytes from MCM-Spegfl/fl mice at 2 weeks post tamoxifen (Figure 7A through 7E). The normalized TT power was reduced to 0.42±0.03 in MCM-Spegfl/fl mice compared with 1.00±0.04 in MCM controls (P<0.001). Given that JPH2 was previously shown to be essential for TT formation and maintenance,5 we measured JPH2 levels in these hearts. Surprisingly, total JPH2 protein levels were unaltered in myocytes from MCM-Spegfl/fl mice (Figure 7F and 7G). Because SPEG is a serine/threonine-kinase, we assessed whether the loss of SPEG altered JPH2 phosphorylation levels. Interestingly, we found that JPH2 phosphorylation was significantly decreased by ≈51% in MCM-Spegfl/fl hearts (P<0.05; Figure 7H and 7I). To confirm that SPEG can phosphorylate JPH2, we transfected JPH2 cDNA in HEK293 cells together with SPEGα or pcDNA3.1 empty vector as a negative control (Online Figure V). Although baseline JPH2 phosphorylation in the absence of SPEG was low, coexpression of SPEG with JPH2 led to a clear increase in JPH2 phosphorylation. Together, our data suggest that defective post-translational modulation of JPH2 by SPEG might play a role in the pathogenesis of HF.

Figure 7.

Figure 7. Loss of transverse tubules (TTs) and junctophilin-2 (JPH2) phosphorylation precedes heart failure in MCM-Spegfl/fl mice. A and B, Representative images of ventricular myocytes isolated from MCM-Spegfl/fl mice or MCM controls 2 weeks post tamoxifen stained with di-8-ANEPPS and zoom (C and D) showing TT structure. E, Quantification of TT power. F, Representative blot of total JPH2 with GAPDH as loading control from mouse heart lysates 2 weeks post tamoxifen and (G) corresponding quantification. H, Representative image of JPH2 phosphoserine (pSer) Western blot normalized to JPH2 from immunoprecipitates from mice 2 weeks post tamoxifen and (I) quantification of phosphoserine normalized to total JPH2. Number in bars=number cells (mice); number hearts. *P<0.05, ***P<0.001. SPEG indicates striated muscle preferentially expressed protein kinase.


This study identified the serine–threonine kinase SPEG as a novel protein within the JMC of cardiac myocytes that have critical functions in regulating ECC. SPEG was found to be a unique binding partner of both the SR Ca2+ release channel RyR2 and JMC structural protein JPH2. In human failing hearts, SPEG levels are downregulated. Using a novel cardiac-specific, inducible Speg knockout mouse model, we established a causal role between SPEG downregulation and HF development coincident with dysregulated SR Ca2+ release and disruption of TTs and the JMC.

The first goal of this study was to identify novel proteins within the JMC, a key subcellular structure in adult cardiac myocytes in which ECC is regulated. To identify new JMC proteins, we performed an unbiased proteomic approach involving IP-MS analysis of 2 key proteins within the JMC, namely the SR Ca2+ release channel RyR2 and structural protein JPH2.17 We used stringent filtering settings to eliminate any potential weak or aspecific interacting proteins. As a result of this approach, we did not find several common binding partners of JPH2 and RyR2 (Online Tables I and II). However, this level of stringency gave us great confidence that SPEG is indeed a specific binding partner of both RyR2 and JPH2 in vivo in the mouse heart. Moreover, the fact that another SR membrane protein SERCA2a did not associate with both RyR2 and JPH2 provides additional evidence for the specificity of our proteomic findings. The IP-MS experiment also revealed that 2 different SPEG isoforms bound to RyR2 and JPH2. These data are consistent with our in vitro coexpression and IP studies in which we found that the N-terminal region of SPEGβ alone binds RyR2 and that SPEGα binds JPH2. Because SPEGβ and SPEGα are the same with the exception of a truncated N terminus in SPEGα, RyR2 can only bind to SPEGβ, whereas JPH2 can bind both SPEGα and SPEGβ. This combination of data obtained in native tissue and a heterologous expression system makes a compelling argument for the specific binding of SPEG to both RyR2 and JPH2.

Our data for the first time demonstrate that SPEG is downregulated by about 80% in patients with nongenetic forms of cardiomyopathy and HF. These findings are consistent with the previous observation that rare SPEG mutations are associated with a genetic disease called centronuclear myopathy, which is often associated with cardiomyopathy.11 Since complete SPEG deficiency is embryonically lethal,10 we developed a novel inducible, tissue-specific Speg knockout model. By crossing the tamoxifen-inducible cardiac myocyte-specific Cre transgenic model (αMHC-mER-Cre-mER) with a floxed Spegfl/fl mouse, we were able to quickly downregulate SPEG levels by 80% in the mouse heart. The fact that there was still some residual SPEG left 2 weeks after tamoxifen treatment may be a testament to the protein turnover dynamics of this protein. Alternatively, it is possible that SPEG expression in nonmyocytes contributes to the residual expression in cardiac tissue. It is interesting that in humans with HF, SPEG levels were also downregulated by ≈80%. Therefore, the data obtained in our MCM-Spegfl/fl mice are not only clinically relevant but also allowed us to establish a causal relationship between induced SPEG downregulation and congestive HF.

Previous studies have focused on the role of SPEG in myocardial development, although no distinction between the function of SPEGα or SPEGβ has been reported to date. Disruption of the Speg gene locus in developing mouse hearts resulted in cardiac myocyte hypertrophy, myofibril degeneration, and impaired cardiac function, leading to increased neonatal mortality.10 It had yet to be determined whether the loss of SPEG in mature myocardium affected cardiac contractility and cardiac myocyte organization. Our study is the first to show that deletion of SPEG in the adult heart has a profound impact on cardiac function and calcium handling. In particular, our results demonstrated that SPEG is required for normal EC coupling. In addition, we found that cardiac myocytes of MCM-Spegfl/fl mice displayed increased Ca2+ spark frequency indicative of increased activity of RyR2, as well as decreased Ca2+ transient amplitudes because of reduced SR Ca2+ stores. Because MCM-Spegfl/fl cardiac myocytes exhibit increased Ca2+ spark frequency without any change in SERCA2a activity, the SR Ca2+ store reduction is likely because of RyR2 leakage, rather than the result of decreased SERCA2a activity. This is consistent with the finding that SPEG does not bind directly to SERCA2a (Figure 1D), indicating that SPEG exerts its effects by regulating RyR2 function in the macromolecular complex.

The mechanism by which SPEG regulates RyR2 function remains largely unknown. SPEGα and SPEGβ, along with obscurin, are members of the myosin light chain kinases that are required for cytoskeletal remodeling in myocytes.9 Because the myosin light chain kinase obscurin subfamily members, including SPEG, contain unique dual serine/threonine kinase domains and multiple protein–protein interaction domains (Figure 1),18 it is possible that enhanced SR Ca2+ leak in SPEG-deficient mice is caused by reduced SPEG-RyR2 binding or decreased SPEG-mediated phosphorylation of RyR2 leading to HF. Indeed, loss of function of Unc-89, an obscurin myosin light chain kinase found in Drosophila and Caenorhabditis elegans, led to RyR dysregulation and contractile dysfunction in C. elegans.18,19 Our data revealed an elevated Ca2+ spark frequency in SPEG-deficient myocytes after the onset of HF, consistent with enhanced RyR2-mediated SR Ca2+ leak, indicating that SPEG is essential for normal RyR2 function. However, before HF, SPEG deficiency did not alter Ca2+ handling or the phosphorylation state of S2808 or S2814, 2 RyR2 phosphorylation sites linked to Ca2+ dysregulation in HF progression.1416 Taken together, SPEG loss seems to have a downstream effect on Ca2+ handling secondary to another primary insult.

Our studies revealed that SPEG also directly binds to JPH2. Previous work from our laboratory showed that JPH2 is critical for TT maintenance, and JPH2 knockdown leads to TT disruption in developing myocardium and in the adult mouse heart.4,5 For this reason, we used TTs as a functional readout for JPH2 function. Our findings showed that SPEG downregulation led to loss of TT regularity and area (Figure 6). Additional experiments showed that this severe disruption of TTs occurred after acute SPEG knockdown before the onset of HF (Figure 7). This TT loss was not because of a decrease in total levels of JPH2 (Figure 7). However, we found that the TT loss was because of altered phosphorylation of JPH2. In line with this theory, previous work showed that JPH2 may be regulated by phosphorylation in HL-1 cells, and loss of JPH2 phosphorylation can lead to cell hypertrophy and Ca2+ dysregulation.20 Here, we demonstrated that SPEG loss before HF leads to decreased JPH2 phosphorylation and TT disruption, suggesting that SPEG indeed regulates JPH2 by phosphorylation. Studies in HEK293 cells confirmed that SPEG overexpression increases JPH2 phosphorylation (Online Figure V). Finally, it is possible that targets of SPEG other than RyR2 or JPH2 may contribute to HF pathogenesis.


Here, we demonstrated that SPEG loss results in hypophosphorylated JPH2, which is a novel mechanism underlying the disruption of TT. This JMC disruption effectively decouples RyR2 from the TTs containing voltage-gated Ca2+ channels, ultimately leading to diastolic Ca2+ leak, impaired ECC, and contractile dysfunction associated with HF. Future studies are warranted to explore whether SPEG within JMCs might represent a novel therapeutic target for HF.

Nonstandard Abbreviations and Acronyms




excitation–contraction coupling


heart failure




junctional membrane complex




mass spectrometry


radioimmunoprecipitation assay


ryanodine receptor type 2


sarcoplasmic endoplasmic reticulum ATPase type 2a


striated muscle preferentially expressed protein kinase


striated muscle preferentially expressed gene (human)


striated muscle preferentially expressed gene (mouse)


sarcoplasmic reticulum


transverse tubule


Western blot


wild type


The Speg mouse strain used for this research project was created from ES cell clone EPD0180_2_A07 obtained from KOMP Repository ( and generated by the Wellcome Trust Sanger Institute.


In September 2016, the average time from submission to first decision for all original research papers submitted to Circulation Research was 12.73 days.

The online-only Data Supplement is available with this article at

Correspondence to Xander H.T. Wehrens, MD, PhD, Baylor College of Medicine, One Baylor Plaza, BCM335, Houston, TX 77030. E-mail


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Novelty and Significance

What Is Known?

  • Excitation–contraction coupling occurs in junctional membrane complexes (JMCs) within cardiac myocytes.

  • Loss of JMC integrity and abnormal intracellular Ca2+ handling are associated with heart failure development.

  • The molecular basis of abnormal sarcoplasmic reticulum Ca2+ handling remains unclear, in part because of a lack of understanding of JMC-targeted proteins.

What New Information Does This Article Contribute?

  • The junctional membrane protein SPEG is downregulated in failing human hearts.

  • Reduced SPEG-mediated phosphorylation of JPH2 causes transverse-tubule disarray, which is an early step during the pathogenesis of heart failure.

JMCs in myocytes are critical microdomains, in which excitation–contraction coupling occurs. Structural and functional disruption of JMCs underlies contractile dysfunction in failing hearts. In our study, we found a novel serine–threonine kinase called SPEG (striated muscle preferentially expressed gene) within the JMC. In human failing hearts, SPEG protein levels were found to be downregulated. Conditional Speg knockout in mouse hearts established a causal link between SPEG downregulation, SPEG-mediated phosphorylation of JPH2, JMC disruption, and heart failure development.


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