Severe dystrophic cardiomyopathy caused by the enteroviral protease 2A-mediated C-terminal dystrophin cleavage fragment.

Enterovirus infection can cause severe cardiomyopathy in humans. The virus-encoded 2A protease is known to cleave the cytoskeletal protein dystrophin. It is unclear, however, whether cardiomyopathy results from the loss of dystrophin or is due to the emergence of a dominant-negative dystrophin cleavage product. We show for the first time that the 2A protease-mediated carboxyl-terminal dystrophin cleavage fragment (CtermDys) is sufficient to cause marked dystrophic cardiomyopathy. The sarcolemma-localized CtermDys fragment caused myocardial fibrosis, heightened susceptibility to myocardial ischemic injury, and increased mortality during cardiac stress testing in vivo. CtermDys cardiomyopathy was more severe than in hearts completely lacking dystrophin. In vivo titration of CtermDys peptide content revealed an inverse relationship between the decay of membrane-bound CtermDys and the restoration of full-length dystrophin at the sarcolemma, in support of a physiologically relevant loss of dystrophin function in this model. CtermDys gene titration and dystrophin replacement studies further established a target threshold of 50% membrane-bound intact dystrophin necessary to prevent mice from CtermDys cardiomyopathy. Conversely, the NtermDys fragment did not compete with dystrophin and had no pathological effect. Thus, CtermDys must be localized to the sarcolemma, with intact dystrophin <50% of normal levels, to exert dominant-negative peptide-dependent cardiomyopathy. These data support a two-hit dominant-negative disease mechanism where membrane-associated CtermDys severs the link to cortical actin and inhibits both full-length dystrophin and compensatory utrophin from binding at the membrane. Therefore, membrane-bound CtermDys is a new potential translational target for virus-mediated cardiomyopathy.

Noncanonical EF-hand motif strategically delays Ca2+ buffering to enhance cardiac performance.

EF-hand proteins are ubiquitous in cell signaling. Parvalbumin (Parv), the archetypal EF-hand protein, is a high-affinity Ca(2+) buffer in many biological systems. Given the centrality of Ca(2+) signaling in health and disease, EF-hand motifs designed to have new biological activities may have widespread utility. Here, an EF-hand motif substitution that had been presumed to destroy EF-hand function, that of glutamine for glutamate at position 12 of the second cation binding loop domain of Parv (ParvE101Q), markedly inverted relative cation affinities: Mg(2+) affinity increased, whereas Ca(2+) affinity decreased, forming a new ultra-delayed Ca(2+) buffer with favorable properties for promoting cardiac relaxation. In therapeutic testing, expression of ParvE101Q fully reversed the severe myocyte intrinsic contractile defect inherent to expression of native Parv and corrected abnormal myocardial relaxation in diastolic dysfunction disease models in vitro and in vivo. Strategic design of new EF-hand motif domains to modulate intracellular Ca(2+) signaling could benefit many biological systems with abnormal Ca(2+) handling, including the diseased heart.

Ex vivo stretch reveals altered mechanical properties of isolated dystrophin-deficient hearts.

Duchenne muscular dystrophy (DMD) is a progressive and fatal disease of muscle wasting caused by loss of the cytoskeletal protein dystrophin. In the heart, DMD results in progressive cardiomyopathy and dilation of the left ventricle through mechanisms that are not fully understood. Previous reports have shown that loss of dystrophin causes sarcolemmal instability and reduced mechanical compliance of isolated cardiac myocytes. To expand upon these findings, here we have subjected the left ventricles of dystrophin-deficient mdx hearts to mechanical stretch. Unexpectedly, isolated mdx hearts showed increased left ventricular (LV) compliance compared to controls during stretch as LV volume was increased above normal end diastolic volume. During LV chamber distention, sarcomere lengths increased similarly in mdx and WT hearts despite greater excursions in volume of mdx hearts. This suggests that the mechanical properties of the intact heart cannot be modeled as a simple extrapolation of findings in single cardiac myocytes. To explain these findings, a model is proposed in which disruption of the dystrophin-glycoprotein complex perturbs cell-extracellular matrix contacts and promotes the apparent slippage of myocytes past each other during LV distension. In comparison, similar increases in LV compliance were obtained in isolated hearts from ß-sarcoglycan-null and laminin-α(2) mutant mice, but not in dysferlin-null mice, suggesting that increased whole-organ compliance in mdx mice is a specific effect of disrupted cell-extracellular matrix contacts and not a general consequence of cardiomyopathy via membrane defect processes. Collectively, these findings suggest a novel and cell-death independent mechanism for the progressive pathological LV dilation that occurs in DMD.

Exercise and muscular dystrophy: implications and analysis of effects on musculoskeletal and cardiovascular systems.

The muscular dystrophies are a heterogeneous collection of progressive, inherited diseases of muscle weakness and degeneration. Although these diseases can vary widely in their etiology and presentation, nearly all muscular dystrophies cause exercise intolerance to some degree. Here, we focus on Duchenne muscular dystrophy (DMD), the most common form of muscular dystrophy, as a paradigm for the effects of muscle disease on exercise capacity. First described in the mid-1800s, DMD is a rapidly progressive and lethal muscular dystrophy caused by mutations in the dystrophin gene. Dystrophin is a membrane-associated cytoskeletal protein, the loss of which causes numerous cellular defects including mechanical instability of the sarcolemma, increased influx of extracellular calcium, and cell signaling defects. Here, we discuss the physiological basis for exercise intolerance in DMD, focusing on the molecular and cellular defects caused by loss of dystrophin and how these manifest as organ-level dysfunction and reduced exercise capacity. The main focus of this article is the defects present in dystrophin-deficient striated muscle. However, discussion regarding the effects of dystrophin loss on other tissues, including vascular smooth muscle is also included. Collectively, the goal of this article is to summarize the current state of knowledge regarding the mechanistic basis for exercise intolerance in DMD, which may serve as an archetype for other muscular dystrophies and diseases of muscle wasting.

Influence of genetic background on ex vivo and in vivo cardiac function in several commonly used inbred mouse strains.

Inbred mouse strains play a critical role in biomedical research. Genetic homogeneity within inbred strains and their general amenability to genetic manipulation have made them an ideal resource for dissecting the physiological function(s) of individual genes. However, the inbreeding that makes inbred mice so useful also results in genetic divergence between them. This genetic divergence is often unaccounted for but may be a confounding factor when comparing studies that have utilized distinct inbred strains. Here, we compared the cardiac function of C57BL/6J mice to seven other commonly used inbred mouse strains: FVB/NJ, DBA/2J, C3H/HeJ, BALB/cJ, 129X1/SvJ, C57BL/10SnJ, and 129S1/SvImJ. The assays used to compare cardiac function were the ex vivo isolated Langendorff heart preparation and in vivo real-time hemodynamic analysis using conductance micromanometry. We report significant strain-dependent differences in cardiac function between C57BL/6J and other commonly used inbred strains. C57BL/6J maintained better cardiac function than most inbred strains after ex vivo ischemia, particularly compared with 129S1/SvImJ, 129X1/SvJ, and C57BL/10SnJ strains. However, during in vivo acute hypoxia 129X1/SvJ and 129S1/SvImJ maintained relatively normal cardiac function, whereas C57BL/6J animals showed dramatic cardiac decompensation. Additionally, C3H/HeJ showed rapid and marked cardiac decompensation in response to esmolol infusion compared with effects of other strains. These findings demonstrate the complex effects of genetic divergence between inbred strains on cardiac function. These results may help inform analysis of gene ablation or transgenic studies and further demonstrate specific quantitative traits that could be useful in discovery of genetic modifiers relevant to cardiac health and disease.

The hydrocarbon seep tubeworm Lamellibrachia luymesi primarily eliminates sulfate and hydrogen ions across its roots to conserve energy and ensure sulfide supply.

Lamellibrachia luymesi (Polychaeta, Siboglinidae) is a deep-sea vestimentiferan tubeworm that forms large bush-like aggregations at hydrocarbon seeps in the Gulf of Mexico. Like all vestimentiferans, L. luymesi obtains its nutrition from sulfide-oxidizing endosymbiotic bacteria, which it houses in an internal organ called the trophosome. This tubeworm has a lifespan of over 170 years and its survival is contingent upon the availability of sulfide during this long period. In sediments underlying L. luymesi aggregations, microbes produce sulfide by coupling sulfate reduction with hydrocarbon oxidation. L. luymesi acquires sulfide from the sediment using a root-like posterior extension of its body that is buried in the sediment. Its symbionts then oxidize the sulfide to produce energy for carbon fixation, and release sulfate and hydrogen ions as byproducts. It is critical for the tubeworm to eliminate these waste ions, and it could do so either across its vascular plume or across its root. In this study, we measured sulfate and proton elimination rates from live L. luymesi and found that they eliminated approximately 85% of the sulfate produced by sulfide oxidation, and approximately 67% of the protons produced by various metabolic processes, across their roots. On the basis of experiments using membrane transport inhibitors, we suggest that L. luymesi has anion exchangers that mediate sulfate elimination coupled with bicarbonate uptake. Roots could be the ideal exchange surface for eliminating sulfate and hydrogen ions for two reasons. First, these ions might be eliminated across the root epithelium using facilitated diffusion, which is energetically economical. Second, sulfate and hydrogen ions are substrates for bacterial sulfate reduction, and supplying these ions into the sediment might help ensure a sustained sulfide supply for L. luymesi over its entire lifespan.