Improvement of cardiac contractile function by peptide-based inhibition of NF-κB in the utrophin/dystrophin-deficient murine model of muscular dystrophy
- Dawn A Delfín†1,
- Ying Xu†2,
- Jennifer M Peterson†3,
- Denis C Guttridge†3,
- Jill A Rafael-Fortney†1 and
- Paul ML Janssen†2Email author
© Delfín et al; licensee BioMed Central Ltd. 2011
Received: 3 February 2011
Accepted: 17 May 2011
Published: 17 May 2011
Duchenne muscular dystrophy (DMD) is an inherited and progressive disease causing striated muscle deterioration. Patients in their twenties generally die from either respiratory or cardiac failure. In order to improve the lifespan and quality of life of DMD patients, it is important to prevent or reverse the progressive loss of contractile function of the heart. Recent studies by our labs have shown that the peptide NBD (Nemo Binding Domain), targeted at blunting Nuclear Factor κB (NF-κB) signaling, reduces inflammation, enhances myofiber regeneration, and improves contractile deficits in the diaphragm in dystrophin-deficient mdx mice.
To assess whether cardiac function in addition to diaphragm function can be improved, we investigated physiological and histological parameters of cardiac muscle in mice deficient for both dystrophin and its homolog utrophin (double knockout = dko) mice treated with NBD peptide. These dko mice show classic pathophysiological hallmarks of heart failure, including myocyte degeneration, an impaired force-frequency response and a severely blunted β-adrenergic response. Cardiac contractile function at baseline and frequencies and pre-loads throughout the in vivo range as well as β-adrenergic reserve was measured in isolated cardiac muscle preparations. In addition, we studied histopathological and inflammatory markers in these mice.
At baseline conditions, active force development in cardiac muscles from NBD treated dko mice was more than double that of vehicle-treated dko mice. NBD treatment also significantly improved frequency-dependent behavior of the muscles. The increase in force in NBD-treated dko muscles to β-adrenergic stimulation was robustly restored compared to vehicle-treated mice. However, histological features, including collagen content and inflammatory markers were not significantly different between NBD-treated and vehicle-treated dko mice.
We conclude that NBD can significantly improve cardiac contractile dysfunction in the dko mouse model of DMD and may thus provide a novel therapeutic treatment for heart failure.
Duchenne muscular dystrophy (DMD) is a degenerating striated muscle disease caused by the absence of the dystrophin protein. Although limb muscle weakness and the loss of ambulation are usually the initial clinical signs of the disease, patients with DMD die from respiratory failure or heart failure. Pertaining to the heart, ninety-five percent of DMD patients develop dilated cardiomyopathy, and over twenty-five percent die from heart failure . These numbers are predicted to grow as prophylactic treatments targeted at maintaining respiratory function improve. This prediction is further supported by the majority of patients with Becker muscular dystrophy (BMD), who have dystrophin mutations that cause a milder skeletal muscle disease, and typically progress to heart failure.
Improving skeletal muscle function has been the central focus of therapeutic development for DMD and BMD. However, therapies targeting only skeletal muscle but not cardiac muscle could potentially aggravate the already present cardiac dysfunction. In order to improve lifespan and quality of life, progressive loss of contractile function in the heart also needs to be prevented or halted. Our recent studies have shown that the inhibition of the NF-κB signaling pathway can improve both limb and diaphragm muscle contractile function in the dystrophin-deficient mdx genotypic mouse model of DMD[5, 6]. This inhibition was achieved by a small, 11 amino-acid peptide named NBD (NEMO Binding Domain) that binds preferentially to the C-terminal regions of the IKKα and IKKβ catalytic components of IκB kinase (IKK) preventing association with the NF-κB essential modulator (NEMO) regulatory subunit and prohibiting downstream NF-κB signaling. The NBD peptide blunted NF-κB signaling, reduced inflammation, enhanced myofiber regeneration, and improved contractile function in the diaphragm muscle in mdx mice[5, 6].
It is interesting to note that of the pharmacological inhibitors tested for improvement of skeletal muscles in animal models of DMD, none, to our knowledge, were directly tested for their effects to improve cardiac function. Recent studies even suggest that the current standard of care pharmacological treatment for DMD, the corticosteroid prednisone, worsens cardiac function in the mdx model[7, 8]. It is not known whether cardiac contractile function can be improved by NBD treatment, but given its ability to dampen both the inflammatory response and stimulate new skeletal muscle growth resulting in improved contractile function, testing the potential of NBD to improve cardiac function in a DMD-related model of cardiomyopathy is warranted. To this end, we focused our current investigation on translating the basic finding of effective NF-κB inhibition into improved cardiac contractile function. We used a model of DMD that is known to have a more severe cardiac dysfunction than the mdx mouse. In this double knock-out (dko) mouse, where both dystrophin and its partially compensating homolog utrophin are both absent, we previously showed that cardiac contractile function at 8 weeks-of-age is severely affected. These relatively young dko mice display the classic pathophysiological hallmarks of end-stage human cardiac failure with a reduced contractile ability, a negative force-frequency relationship, and a severely blunted β-adrenergic response. In addition these dko mice show cardiac muscle degeneration and by 10 weeks of age they have replacement of damaged cardiomyocytes with fibrotic scars, similar to both DMD patients  and the larger heart failure population[15, 16]. Therefore, improvement in cardiac function in these mice would have possible therapeutic implications not only for cardiomyopathy in the muscular dystrophies, but also possibly for the much larger population of heart failure patients suffering from cardiac contractile dysfunction.
In this study, to completely assess functional aspects of NBD treatment, we investigated both the baseline contractile function of the myocardium and the regulation of contractility in the dko mice. We assessed length-dependent activation, frequency-dependent activation, and β-adrenergic stimulation in isolated dko cardiac papillary muscles treated with NBD peptide or vehicle. The results indicate that NBD can significantly improve cardiac contractile dysfunction in this model of muscular dystrophy cardiomyopathy.
Utrophin/dystrophin-deficient double knockout (utrn -/- ;mdx, dko) offspring were born at an approximately 1:4 ratio from matings between utrn +/- ;mdx mice. Offspring were genotyped shortly after birth as described previously and both male and female dko mice were used for treatment and control groups. Experimental protocols involving mice were approved by the Institutional Animal Care and Use Committee at The Ohio State University.
Peptide synthesis of NBD was the same as described previously.
Treatment with NBD was initiated when mice were less than one week of age. NBD diluted in 10% DMSO in phosphate buffered saline (PBS) was delivered by intraperitoneal injection 3 times weekly until the mice were 8 weeks-of-age. Because mice were actively growing during the first half of the treatment time and as adults their weights are variable, dko mice were weighed prior to each injection until 4 weeks of age and then once each week thereafter to achieve the desired 10 mg/kg peptide dosage. In previous studies scrambled peptide sequences showed no functional differences versus vehicle alone[5, 17]. The control group for this study consisted of dko mice that were injected on the same schedule with an equal volume of the vehicle (10% DMSO in PBS).
EMSA and Western Blotting
EMSA and western analyses were performed as previously described for skeletal muscle tissue [5, 6, 18] from cardiac ventricular tissue from vehicle or NBD treated dko mice. Heart tissue was homogenized and cytoplasmic extracts were prepared using an extraction buffer with standard protease inhibitors. After incubation and mild centrifugation, nuclear extracts were further isolated by using two pellet volumes of extraction buffer and standard protease inhibitors. Nuclear pellets were resuspended by vortexing and transferred to fresh tubes for use in EMSA analysis. These prepared nuclear extracts were either incubated with a radioactive oligonucleotide containing a consensus NF-∣B binding site and fractionated on a 5% non-denaturing polyacrylamide gel (EMSA) or used in a western blot and probed against p65.
Assessment of contractile physiology
At the end of the treatment regimen, contractile function of cardiac muscle tissue was assessed in vitro, as previously described[10, 19, 20]. Briefly, under deep anesthesia, hearts were rapidly removed, and flushed with a Krebs-Henseleit solution. The right ventricle was opened, and small papillary muscles were dissected under a stereo microscope. The muscles were mounted in an experimental chamber, superfused with Krebs-Henseleit solution, containing 1.5 mM Ca2+, at 37°C. Muscles were electrically stimulated to twitch contract, and force of contraction was recorded. First, after the muscle had equilibrated in the set-up, muscle length was increased until a further increase in length no longer resulted in an increase in active twitch developed peak force. This length was then considered optimal length. Because the heart regulates contractile force through several physiological mechanisms, it is important not only to assess baseline contractile parameters, but also the response to normal physiological regulatory mechanisms. Therefore, we assessed the main three mechanisms used by the heart to regulate contractile strength: length-dependent behavior, frequency-dependent stimulation, and β-adrenergic stimulation. After assessment of baseline contractile parameters, at a stimulation frequency of 4 Hz, these three regulatory responses were assessed in each muscle, using protocols described previously[10, 19]. The experimenters were blinded to the treatment of the mice. If more than 1 muscle was measured per mouse, data were averaged to reduce variability. N-numbers reported reflect numbers of mice studied.
After cardiac muscle samples for physiological analyses were removed, the remaining heart tissue was frozen in Optimal Cutting Temperature (O.C.T.) medium (Tissue-Tek, Torrance, CA) on liquid nitrogen-cooled isopentane. Serial cryosections (8 μm) were cut from the tissue blocks and used for the following staining procedures. For viewing of gross histology, sections were fixed in 100% ethanol and then stained with hematoxylin and eosin using standard procedures. For specific detection of fibrosis, fibroblasts, and immune cells in regions of cardiac damage, immunofluorescence was performed on serial cryosections. Unfixed cryosections were equilibrated in KPBS (16.4 mM K2HPO4, 3.6 mM KH2PO4, 160 mM NaCl) for 5 minutes then blocked with KPBS + 1% gelatin for 15 minutes. Slides were washed with KPBS + 0.2% gelatin (KPBSG), then incubated for two hours with primary antibodies, which were diluted in KPBSG + 1% normal goat serum, against collagen I (Abcam, Cambridge, MA, ab292 rabbit polyclonal) at 1:200, ER-TR7 (Abcam ab51824 rat monoclonal) at 1:100, or CD45 (BD Pharmingen, Franklin Lakes, NJ, 550539 rat monoclonal) at 1:50. Slides were washed and then incubated for one hour with Cy3-conjugated goat secondary antibodies against rabbit IgG (Jackson Immuno Research, West Grove, PA,111-165-144) or rat IgG (Jackson Immuno Research 712-165-153), diluted 1:100 in KPBSG + 1% normal goat serum, for detection of bound primary antibodies. Slides were again washed, and then mounted in Vectashield (Vector Labs, Burlingame, CA) containing 2 μg/ml DAPI (Sigma, Saint Louis, MO) to stain nuclei. Fluorescence was viewed with a Nikon Eclipse 800 microscope (Nikon Corporation, Tokyo, Japan) and imaged with a SPOT-RTslider digital camera and SPOT software (Diagnostic Instruments, Inc., Sterling Heights, MI). Control experiments using secondary antibodies only revealed no staining.
Contractile forces were analyzed using unpaired t-tests or ANOVA, followed by post-hoc tests where applicable. A two-tailed P value of < 0.05 was considered significant.
Lastly, we investigated whether NBD treatment of dko mice resulted in an improvement in cardiac histopathological features of this model. Between eight and ten weeks-of-age, dko mice display myocardial damage followed by fibrotic scarring in damaged regions. Despite the robust improvement in contractile function resulting from NBD-treatment, and the well-documented role of NF-κB in inflammation, histopathological features of the dko myocardium were not markedly improved by NBD treatment.
Cardiac contractile dysfunction is one of the leading causes of death in DMD. Clinical treatment of this debilitating aspect of DMD is paramount in extending both life-span and quality of life. In this study we showed that a peptide referred to as NBD which blunts NF-κB signaling, can restore cardiac contractile dysfunction in a mouse model of DMD. Not only did NBD treatment increase contractile force substantially, it also improved key governing mechanisms of contractile force that are typically impaired in patients with heart failure including force-frequency behavior and the response to β-adrenergic stimulation[11, 12, 25].
The increased contractile strength was likely not a direct effect of altered histology of the myocardium. We observed no significant reduction in fibrosis in the dko myocardium upon treatment with NBD. However, we cannot at this point exclude that local improvements in the histology of papillary muscles may play a role. Most of the area of the right ventricle and septum where the muscles were excised is unsuitable for histological analysis due to the dissection. The muscles used for physiological force measurements, after experimentation, are also not suitable for histological analysis and subsequent correlative analysis. Thus, we cannot show a potential histological change in the preparations where function was actually assessed. However, given the widespread fibrosis that was still clearly present in the remaining ventricular tissue after NBD treatment, a local improvement of histopathology being primarily responsible for the improved function is quite unlikely. At present, and well beyond the scope of this proof-of-principle study, we can only speculate about the underlying molecular events that ultimately result in an improvement of contractile function.
The underlying cause of weakened contractile performance of the end-stage heart failing myocardium is often independent of the originating cause of heart failure in a patient or animal model. Impaired calcium handling is a central finding in end-stage heart failure, and this impaired calcium handling correlates with the blunting, or even loss, of frequency-dependent activation. In human heart failure, the normal positive force frequency response is typically severely blunted, or even becomes negative, and is a hallmark of the phenotypic dysfunction[11, 24, 25]. In normal, healthy mice, when frequency of contraction is increased, the force development of the muscle is generally slightly increased or at least does not show a major decrease, while relaxation is always faster. However, in mice with cardiac dysfunction, such as the dko mouse used in this study, the force-frequency relationship is clearly negative. We show that NBD treatment not only increases contractile strength of dko myocardium, but it also significantly improved the force-frequency relationship. This response was no longer largely negative, and even reverted to positive at the lower end of the frequency range, resembling the frequency-dependent behavior typically found in healthy mice. The restoration of a normal force-frequency response is thus indirect evidence that calcium handling improvement may be the major underlying factor in the functional improvement of dko myocardium after NBD treatment.
NF-κB and calcium ions are both multifaceted signaling molecules and interactions between calcium ion concentration and NF-κB have been documented. For instance, in smooth muscle, NF-κB is negatively impacted by calcium channels, and thus inhibition by NBD could potentially upregulate these calcium channels, improving function by facilitating calcium influx. Also, inhibition of NF-κB has been shown to be able to alleviate sarcoplasmic reticulum stress, and interact with levels of the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), which is responsible for the uptake of calcium ions from the cytoplasm. NF-κB in skeletal muscle has been shown to modulate expression of nitric oxide synthase (NOS) isoforms, which play an important role in maintaining cardiovascular homeostasis mainly via calcium handling. Lastly, a recent report by Panama and colleagues  showed that NF-κB downregulates the transient outward potassium current in the heart, further providing evidence for a role of NF-κB regulated processes in excitation-contraction (EC)-coupling. Therefore, although we have no direct conclusive evidence at this stage, NBD may improve contractile function in dko myocardium via improvement in EC-coupling/calcium handling, rather than via a prevention of cardiac histologically-detectable damage. Dystrophic skeletal muscle function can be improved by low levels of dystrophin in absence of histopathological improvement. Therefore, a similar improvement of function of non-fibrotic dystrophic myocardium may account for the results of our study. Further targeted studies are required to elucidate possible mechanisms and could include electrophysiological and heamodynamic assessments[33, 34], as well as intracellular calcium handling. Any therapeutic strategy involving NBD may require a combinatorial approach with a factor that would prevent cardiac damage
In addition to reduced contractility and a negative force-frequency response, it is well known that both in patients with heart failure, as well as in many animal models of cardiac dysfunction, the physiological response to β-adrenergic stimulation is severely blunted. In untreated dko myocardium, this blunted β-adrenergic response is typically observed, and is severe, and in the present study we found that NBD treatment significantly improves this response. The main underlying molecular level events that lead to increased contractility after β-adrenergic stimulation may again be found in the enhancement of the intracellular calcium transient. Thus, the same mechanism responsible for the improved force-frequency response could be the main factor for improvement of this β-response.
In this study we show that inhibition of NF-κB using the small peptide inhibitor NBD improves contractile force, improves the force-frequency relationship, and restores the response to β-adrenergic stimulation in the well-established murine model for cardiac dysfunction associated with DMD. Since we have demonstrated a therapeutic effect of NBD on both skeletal and cardiac muscle (this study), NBD peptide treatment may be a realistic treatment option for this debilitating disease. Moreover, because the dko mouse model recapitulates many of the contractile phenotypes found in the majority of patients with end-stage failure stemming from a variety of etiologies, NBD treatment may be useful beyond the field of muscular dystrophy.
This study was supported by a grant from the National Institutes of Health U01 NS058451 (To DG, PMLJ, and JRF), K02 HL08357 (to PMLJ), T32 HL098039 (support to DAD), as well as support from the Muscular Dystrophy Association (to DCG and JMP) and the American Heart Association (EIA 0740040N to PMLJ). The authors declare that they have no competing interests.
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