Benjamin D. Canan

A genetic model of amyotrophic lateral sclerosis in zebrafish displays phenotypic hallmarks of motoneuron disease.

SUMMARY Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that, for ∼80% of patients, is fatal within five years of diagnosis. To better understand ALS, animal models have been essential; however, only rodent models of ALS exhibit the major hallmarks of the disease. Here, we report the generation of transgenic zebrafish overexpressing mutant Sod1. The construct used to generate these lines contained the zebrafish sod1 gene and ∼16 kb of flanking sequences. We generated lines expressing the G93R mutation, as well as lines expressing wild-type Sod1. Focusing on two G93R lines, we found that they displayed the major phenotypes of ALS. Changes at the neuromuscular junction were observed at larval and adult stages. In adulthood the G93R mutants exhibited decreased endurance in a swim tunnel test. An analysis of muscle revealed normal muscle force, however, at the end stage the fish exhibited motoneuron loss, muscle atrophy, paralysis and premature death. These phenotypes were more severe in lines expressing higher levels of mutant Sod1 and were absent in lines overexpressing wild-type Sod1. Thus, we have generated a vertebrate model of ALS to complement existing mammal models.

IKKα and alternative NF-κB regulate PGC-1β to promote oxidative muscle metabolism

Alternative NF-κB signaling modulates the activity of PGC-1β to promote oxidative metabolism in skeletal muscle.

Micro-dystrophin and follistatin co-delivery restores muscle function in aged DMD model

Pharmacologic strategies have provided modest improvement in the devastating muscle-wasting disease, Duchenne muscular dystrophy (DMD). Pre-clinical gene therapy studies have shown promise in the mdx mouse model; however, studies conducted after disease onset fall short of fully correcting muscle strength or protecting against contraction-induced injury. Here we examine the treatment effect on muscle physiology in aged dystrophic mice with significant disease pathology by combining two promising therapies: micro-dystrophin gene replacement and muscle enhancement with follistatin, a potent myostatin inhibitor. Individual treatments with micro-dystrophin and follistatin demonstrated marked improvement in mdx mice but were insufficient to fully restore muscle strength and response to injury to wild-type levels. Strikingly, when combined, micro-dystrophin/follistatin treatment restored force generation and conferred resistance to contraction-induced injury in aged mdx mice. Pre-clinical studies with miniature dystrophins have failed to demonstrate full correction of the physiological defects seen in mdx mice. Importantly, the addition of a muscle enhancement strategy with delivery of follistatin in combination with micro-dystrophin gene therapy completely restored resistance to eccentric contraction-induced injury and improved force. Eccentric contraction-induced injury is a pre-clinical parameter relevant to the exercise induced injury that occurs in DMD patients, and herein, we demonstrate compelling evidence for the therapeutic potential of micro-dystrophin/follistatin combinatorial therapy.

mdx5cv Mice Manifest More Severe Muscle Dysfunction and Diaphragm Force Deficits than Do mdx Mice

Duchenne muscular dystrophy (DMD) is characterized by progressive skeletal muscle dysfunction leading to premature death by the third decade of life. The mdx mouse, the most widely used animal model of DMD, has been extremely useful to study disease mechanisms and to screen new therapeutics. However, unlike patients with DMD, mdx mice have a very mild motor function deficit, posing significant limitations for its use as a platform to assess the impact of treatments on motor function. It has been suggested that an mdx variant, the mdx(5cv) mouse, might be more severely affected. Here, we compared the motor activity, histopathology, and individual muscle force measurements of mdx and mdx(⁵cv) mice. Our study revealed that mdx(⁵cv) mice showed more severe exercise-induced fatigue, Rotarod performance deficits, and gait anomalies than mdx mice and that these deficits began at a younger age. Muscle force studies showed more severe strength deficits in the diaphragm of mdx(⁵cv) mice compared to mdx mice, but similar force generation in the extensor digitorum longus. Muscle histology was similar between the two strains. Differences in genetic background (genetic modifiers) probably account for these functional differences between mdx strains. Overall, our findings indicate that the mdx and mdx(⁵cv) mouse models of DMD are not interchangeable and identify the mdx(⁵cv) mouse as a valuable platform for preclinical studies that require assessment of muscle function in live animals.

In Vivo Genome Editing Restores Dystrophin Expression and Cardiac Function in Dystrophic Mice

Rationale: Duchenne muscular dystrophy is a severe inherited form of muscular dystrophy caused by mutations in the reading frame of the dystrophin gene disrupting its protein expression. Dystrophic cardiomyopathy is a leading cause of death in Duchenne muscular dystrophy patients, and currently no effective treatment exists to halt its progression. Recent advancement in genome editing technologies offers a promising therapeutic approach in restoring dystrophin protein expression. However, the impact of this approach on Duchenne muscular dystrophy cardiac function has yet to be evaluated. Therefore, we assessed the therapeutic efficacy of CRISPR (clustered regularly interspaced short palindromic repeats)-mediated genome editing on dystrophin expression and cardiac function in mdx/Utr+/− mice after a single systemic delivery of recombinant adeno-associated virus. Objective: To examine the efficiency and physiological impact of CRISPR-mediated genome editing on cardiac dystrophin expression and function in dystrophic mice. Methods and Results: Here, we packaged SaCas9 (clustered regularly interspaced short palindromic repeat–associated 9 from Staphylococcus aureus) and guide RNA constructs into an adeno-associated virus vector and systemically delivered them to mdx/Utr+/− neonates. We showed that CRIPSR-mediated genome editing efficiently excised the mutant exon 23 in dystrophic mice, and immunofluorescence data supported the restoration of dystrophin protein expression in dystrophic cardiac muscles to a level approaching 40%. Moreover, there was a noted restoration in the architecture of cardiac muscle fibers and a reduction in the extent of fibrosis in dystrophin-deficient hearts. The contractility of cardiac papillary muscles was also restored in CRISPR-edited cardiac muscles compared with untreated controls. Furthermore, our targeted deep sequencing results confirmed that our adeno-associated virus-CRISPR/Cas9 strategy was very efficient in deleting the ≈23 kb of intervening genomic sequences. Conclusions: This study provides evidence for using CRISPR-based genome editing as a potential therapeutic approach for restoring dystrophic cardiomyopathy structurally and functionally.

The Frank-Starling mechanism involves deceleration of cross-bridge kinetics and is preserved in failing human right ventricular myocardium

Cross-bridge cycling rate is an important determinant of cardiac output, and its alteration can potentially contribute to reduced output in heart failure patients. Additionally, animal studies suggest that this rate can be regulated by muscle length. The purpose of this study was to investigate cross-bridge cycling rate and its regulation by muscle length under near-physiological conditions in intact right ventricular muscles of nonfailing and failing human hearts. We acquired freshly explanted nonfailing (n = 9) and failing (n = 10) human hearts. All experiments were performed on intact right ventricular cardiac trabeculae (n = 40) at physiological temperature and near the normal heart rate range. The failing myocardium showed the typical heart failure phenotype: a negative force-frequency relationship and β-adrenergic desensitization (P < 0.05), indicating the expected pathological myocardium in the right ventricles. We found that there exists a length-dependent regulation of cross-bridge cycling kinetics in human myocardium. Decreasing muscle length accelerated the rate of cross-bridge reattachment (ktr) in both nonfailing and failing myocardium (P < 0.05) equally; there were no major differences between nonfailing and failing myocardium at each respective length (P > 0.05), indicating that this regulatory mechanism is preserved in heart failure. Length-dependent assessment of twitch kinetics mirrored these findings; normalized dF/dt slowed down with increasing length of the muscle and was virtually identical in diseased tissue. This study shows for the first time that muscle length regulates cross-bridge kinetics in human myocardium under near-physiological conditions and that those kinetics are preserved in the right ventricular tissues of heart failure patients.

Sustaining Cardiac Claudin-5 Levels Prevents Functional Hallmarks of Cardiomyopathy in a Muscular Dystrophy Mouse Model

Identification of new molecular targets in heart failure could ultimately have a substantial positive impact on both the health and financial aspects of treating the large heart failure population. We originally identified reduced levels of the cell junction protein claudin-5 specifically in heart in the dystrophin/utrophin-deficient (Dmd(mdx);Utrn(-/-)) mouse model of muscular dystrophy and cardiomyopathy, which demonstrates physiological hallmarks of heart failure. We then showed that at least 60% of cardiac explant samples from patients with heart failure resulting from diverse etiologies also have reduced claudin-5 levels. These claudin-5 reductions were independent of changes in other cell junction proteins previously linked to heart failure. The goal of this study was to determine whether sustaining claudin-5 levels is sufficient to prevent the onset of histological and functional indicators of heart failure. Here, we show the proof-of-concept rescue experiment in the Dmd(mdx);Utrn(-/-) model, in which claudin-5 reductions were originally identified. Expression of claudin-5 4 weeks after a single administration of recombinant adeno-associated virus (rAAV) containing a claudin-5 expression cassette prevented the onset of physiological hallmarks of cardiomyopathy and improved histological signs of cardiac damage. This experiment demonstrates that claudin-5 may represent a novel treatment target for prevention of heart failure.

Claudin-5 levels are reduced from multiple cell types in human failing hearts and are associated with mislocalization of ephrin-B1

Claudin-5 is transcriptionally downregulated resulting in dramatically reduced protein levels in human heart failure. Studies in mice have demonstrated that reduced claudin-5 levels occur prior to cardiac damage and far before reduced whole heart function. Therefore, claudin-5 may be a useful early therapeutic target for human heart failure. However, the cell types in which claudin-5 is localized in human heart and from which claudin-5 is reduced in heart failure is not known. The recent identification of claudin-5s interaction with ephrin-B1 in mouse hearts has also not been investigated in non-failing or failing human hearts. In this study we collected human left ventricular mid-myocardium histological samples from 7 non-failing hearts and 16 end-stage failing hearts. Immunoblots demonstrate severe reductions of claudin-5 protein in 14 of 16 failing hearts compared to non-failing controls. Claudin-5 was observed to localize to cardiomyocytes, endothelial cells, and a subset of fibroblasts in non-failing human heart sections. In isolated cardiomyocytes, the transmembrane claudin-5 protein localized in longitudinal striations in lateral membranes. In failing heart, both cardiomyocyte and endothelial claudin-5 localization was severely reduced, but claudin-5 remained in fibroblasts. Absence of claudin-5 staining also correlated with the reduction of the endothelial cell marker CD31. Ephrin-B1 localization, but not protein levels, was altered in failing hearts supporting that claudin-5 is required for ephrin-B1 localization. These data support that loss of claudin-5 in cardiomyocytes and endothelial cells is prevalent in human heart failure. Investigating claudin-5/ephrin-B1 protein complexes and gene regulation may lead to novel therapies.

The force-temperature relationship in healthy and dystrophic mouse diaphragm; implications for translational study design

In the field of muscular dystrophy, striated muscle function is often assessed in vitro in dystrophin-deficient mdx mice in order to test the impact of a potential treatment strategy. Although many past studies have assessed diaphragm contractile function at or near room temperature, the diaphragm performs in vivo at 37°C. To improve translation of bench-top results to possible clinical application, we studied temperature-dependence of contractile performance in wild-type (C57BL/10) and mdx muscle strips at temperatures from 25°C to 37°C. Maximal tetanic force in wild-type muscles was higher at 37°C (198 ± 11 vs. 155 ± 9 mN/mm2 at 25°C), while the difference between wild-type and mdx was extremely similar: wild-type muscles produced 45.9% and 45.1% more force at 25°C and 37°C respectively. At 37°C twitch contraction kinetics and 50% rise time to tetanic plateau were slower in mdx diaphragm. A fatigue/injury protocol indicated 2-fold fatigue/contraction-induced force deficit in mdx muscles. We conclude that assessment of diaphragm muscle strips can be reliably and reproducibly performed at 37°C.

Effects of zacopride, a moderate IK1 channel agonist, on triggered arrhythmia and contractility in human ventricular myocardium

Graphical abstract Figure. No caption available. ABSTRACT Ventricular tachycardia is the leading cause of sudden arrhythmic death in the U.S. Recently, the moderate IK1 channel activator, zacopride, was shown to suppress triggered ventricular tachycardia in rats. Nonetheless, concerns were raised about the possibility of pro‐arrhythmic activity after IK1 channel stimulation based on the promising anti‐arrhythmic strategy of IK1 blockade in other animal models. Therefore, the goal of the current study was to investigate the ex‐vivo effects of zacopride on triggered arrhythmia and contractility in ventricular human myocardium in order to validate data that was solely obtained from animal models. Application of 100 nmol/L isoproterenol and 0.5 mmol/L caffeine led to triggered arrhythmia in isolated cardiac muscles from non‐failing and end‐stage failing hearts. However, the occurrence of arrhythmia in muscles of non‐failing hearts was markedly higher than those of end‐stage failing hearts. Interestingly, zacopride eliminated the ex‐vivo triggered arrhythmia in these muscles of non‐failing and failing hearts in a concentration‐dependent manner, with an effective IC50 in the range of 28–40 &mgr;mol/L. Conversely, in the absence of isoproterenol/caffeine, zacopride led to a negative inotropic effect in a concentration‐dependent manner. Reduced cardiac contraction was clearly observed at high zacopride concentration of 200 &mgr;mol/L, along with the occurrence of contractile alternans in muscles of non‐failing and failing hearts. Zacopride shows promising antiarrhythmic effects against triggered arrhythmia in human ventricular myocardium. However, in the absence of Ca2+ overload/arrhythmia, zacopride, albeit at high concentrations, decreases the force of contraction and increases the likelihood of occurrence of contractile alternans, which may predispose the heart to contractile dysfunction and/or arrhythmia. Overall, our results represent a key step in translating this drug from the benchtop to the bedside in the research area.