A morpholino oligomer therapy regime that restores mitochondrial function and prevents mdx cardiomyopathy

Current clinical trials demonstrate Duchenne muscular dystrophy (DMD) patients receiving phosphorodiamidate morpholino oligomer (PMO) therapy exhibit improved ambulation and stable pulmonary function; however, cardiac abnormalities remain. Utilizing the same PMO chemistry as current clinical trials, we have identified a non-toxic PMO treatment regimen that restores metabolic activity and prevents DMD cardiomyopathy. We propose that a treatment regimen of this nature may have the potential to significantly improve morbidity and mortality from DMD by improving ambulation, stabilizing pulmonary function, and preventing the development of cardiomyopathy

Author Correction: Identification of a novel cAMP dependent protein kinase A phosphorylation site on the human cardiac calcium channel

The original version of this Article contained a typographical error in the spelling of the author A. Harvey Millar, which was incorrectly given as Harvey A. Millar. This has now been corrected in the PDF and HTML versions of the Article and in the Supplementary Information

Fidelity and coordination of mitochondrial protein synthesis in health and disease

The evolutionary acquisition of mitochondria has given rise to the diversity of eukaryotic life. Mitochondria have retained their ancestral α-proteobacterial traits through the maintenance of double membranes and their own circular genome. Their genome varies in size from very large in plants to the smallest in animals and their parasites. The mitochondrial genome encodes essential genes for protein synthesis and has to coordinate its expression with the nuclear genome from which it sources most of the proteins required for mitochondrial biogenesis and function. The mitochondrial protein synthesis machinery is unique because it is encoded by both the nuclear and mitochondrial genomes thereby requiring tight regulation to produce the respiratory complexes that drive oxidative phosphorylation for energy production. The fidelity and coordination of mitochondrial protein synthesis are essential for ATP production. Here we compare and contrast the mitochondrial translation mechanisms in mammals and fungi to bacteria and reveal that their diverse regulation can have unusual impacts on the health and disease of these organisms. We highlight that in mammals the rate of protein synthesis is more important than the fidelity of translation, enabling coordinated biogenesis of the mitochondrial respiratory chain with respiratory chain proteins synthesised by cytoplasmic ribosomes. Changes in mitochondrial protein fidelity can trigger the activation of the diverse cellular signalling networks in fungi and mammals to combat dysfunction in energy conservation. The physiological consequences of altered fidelity of protein synthesis can range from liver regeneration to the onset and development of cardiomyopathy. (Figure presented.)

Impaired functional communication between the L-type calcium channel and mitochondria contributes to metabolic inhibition in the mdx heart

Duchenne muscular dystrophy is a fatal X-linked disease characterized by the absence of dystrophin. Approximately 20% of boys will die of dilated cardiomyopathy that is associated with cytoskeletal protein disarray, contractile dysfunction, and reduced energy production. However, the mechanisms for altered energy metabolism are not yet fully clarified. Calcium influx through the L-type Ca2+ channel is critical for maintaining cardiac excitation and contraction. The L-type Ca2+ channel also regulates mitochondrial function and metabolic activity via transmission of movement of the auxiliary beta subunit through intermediate filament proteins. Here, we find that activation of the L-type Ca2+ channel is unable to induce increases in mitochondrial membrane potential and metabolic activity in intact cardiac myocytes from the murine model of Duchenne muscular dystrophy (mdx) despite robust increases recorded in wt myocytes. Treatment of mdx mice with morpholino oligomers to induce exon skipping of dystrophin exon 23 (that results in functional dystrophin accumulation) or application of a peptide that resulted in block of voltage-dependent anion channel (VDAC) “rescued” mitochondrial membrane potential and metabolic activity in mdx myocytes. The mitochondrial VDAC coimmunoprecipitated with the L-type Ca2+ channel. We conclude that the absence of dystrophin in the mdx ventricular myocyte leads to impaired functional communication between the L-type Ca2+ channel and mitochondrial VDAC. This appears to contribute to metabolic inhibition. These findings provide new mechanistic and functional insight into cardiomyopathy associated with Duchenne muscular dystrophy

A common genetic variant of a mitochondrial RNA processing enzyme predisposes to insulin resistance

Mitochondrial energy metabolism plays an important role in the pathophysiology of insulin resistance. Recently, a missense N437S variant was identified in the MRPP3 gene, which encodes a mitochondrial RNA processing enzyme within the RNase P complex, with predicted impact on metabolism. We used CRISPR-Cas9 genome editing to introduce this variant into the mouse Mrpp3 gene and show that the variant causes insulin resistance on a high-fat diet. The variant did not influence mitochondrial gene expression markedly, but instead, it reduced mitochondrial calcium that lowered insulin release from the pancreatic islet β cells of the Mrpp3 variant mice. Reduced insulin secretion resulted in lower insulin levels that contributed to imbalanced metabolism and liver steatosis in the Mrpp3 variant mice on a high-fat diet. Our findings reveal that the MRPP3 variant may be a predisposing factor to insulin resistance and metabolic disease in the human population

Identification of a novel cAMP dependent protein kinase A phosphorylation site on the human cardiac calcium channel

The “Fight or Flight” response is elicited by extrinsic stress and is necessary in many species for survival. The response involves activation of the β-adrenergic signalling pathway. Surprisingly the mechanisms have remained unresolved. Calcium influx through the cardiac L-type Ca2+ channel (Cav1.2) is absolutely required. Here we identify the functionally relevant site for PKA phosphorylation on the human cardiac L-type Ca2+ channel pore forming α1 subunit using a novel approach. We used a cell free system where we could assess direct effects of PKA on human purified channel protein function reconstituted in proteoliposomes. In addition to assessing open probability of channel protein we used semi-quantitative fluorescent phosphoprotein detection and MS/MS mass spectrometry analysis to demonstrate the PKA specificity of the site. Robust increases in frequency of channel openings were recorded after phosphorylation of the long and short N terminal isoforms and the channel protein with C terminus truncated at aa1504. A protein kinase A anchoring protein (AKAP) was not required. We find the novel PKA phosphorylation site at Ser1458 is in close proximity to the Repeat IV S6 region and induces a conformational change in the channel protein that is necessary and sufficient for increased calcium influx through the channe

Identifying the site of the source of reactive oxygen species within the mitochondria after transient exposure of cardiac myocytes to hydrogen peroxide

Oxidative stress is a feature of cardiovascular disease. Hydrogen peroxide (H2O2) can act as a signaling molecule to mediate cardiovascular pathology. We have previously shown that transient exposure of adult guinea pig ventricular myocytes to H2O2 leads to further production of reactive oxygen species (ROS) from the mitochondria. We have demonstrated that exposure of myocytes to 30μM H2O2 for 5 min then 10U/ml catalase for 5 min to degrade the H2O2 caused a 65.4±8.4% further increase in superoxide by the mitochondria (n=47). We tested whether transient exposure to H2O2 altered protein synthesis in the myocytes. Exposure of myocytes to 30μM H2O2 for 5 min followed by 10U/ml catalase for 5 min caused a 2-fold increase in protein synthesis measured as 3H-Leucine incorporation (n=10). This suggests that a transient exposure to H2O2 may be sufficient to induce cardiac hypertrophy. We now wish to identify the site of ROS production in the mitochondria. Superoxide was assessed with the fluorescent indicator dihydroethidium (DHE). Exposing myocytes to 1μM DPI, which binds prior to the ROS generation site of complex I, followed by transient exposure to H2O2 resulted in complete attenuation of the increase in DHE signal after exposure to H2O2. Exposing myocytes to 1μM rotenone, which binds after the ROS generation site of complex I, followed by transient exposure to H2O2 resulted in a 45% reduction in the increase in DHE signal after exposure to H2O2. These data suggest the source of ROS production is distal to complex I. Identifying the site of production of ROS may represent a possible therapeutic target to prevent the development of cardiac hypertrophy associated with a transient exposure to H2O2

The cardiac L-type calcium channel alpha subunit is a target for direct redox modification during oxidative stress-the role of cysteine residues in the alpha interacting domain

Cardiovascular disease is the leading cause of death in the Western world. The incidence of cardiovascular disease is predicted to further rise with the increase in obesity and diabetes and with the aging population. Even though the survival rate from ischaemic heart disease has improved over the past 30 years, many patients progress to a chronic pathological condition, known as cardiac hypertrophy that is associated with an increase in morbidity and mortality. Reactive oxygen species (ROS) and calcium play an essential role in mediating cardiac hypertrophy. The L-type calcium channel is the main route for calcium influx into cardiac myocytes. There is now good evidence for a direct role for the L-type calcium channel in the development of cardiac hypertrophy. Cysteines on the channel are targets for redox modification and glutathionylation of the channel can modulate the function of the channel protein leading to the onset of pathology. The cysteine responsible for modification of L-type calcium channel function has now been identified. Detailed understanding of the role of cysteines as possible targets during oxidative stress may assist in designing therapy to prevent the development of hypertrophy and heart failure

PMO-mediated dystrophin exon 23 skipping restores nitochondrial function in the MDX heart

Approximately 20% of boys with Duchenne Muscular Dystrophy will die of dilated cardiomyopathy. The cardiomyopathy is characterised by disrupted structure and function of cardiac muscle cells and reduced energy production. However, the mechanisms responsible for the altered energy metabolism have been poorly understood. We have previously sought to identify the mechanisms for metabolic inhibition in mdx mouse cardiomyopathy. Calcium influx through the L-type Ca2+ channel (also known as the dihydropyridine receptor) in cardiac myocytes is essential for contraction. Calcium is also important for the regulation of mitochondrial function and production of ATP that is required to meet the energy demands of the heart. We have shown that the L-type Ca2+ channel can regulate mitochondrial function and metabolic activity in cardiac myocytes. In mdx heart, the communication between the Ltype Ca2+ channel and the mitochondria is altered as a result of disruption of the cytoskeletal architecture. This contributes to metabolic inhibition in the mdx heart. We demonstrate that treatment of mdx mice with a phophorodiamidate morpholino oligomer, designed to induce skipping of exon 23, “restored” the increase in mitochondrial membrane potential in mdx myocytes after activation of the L-type Ca2+ channel with the dihydropyridine. These results confirm that metabolic inhibition occurs as a result of the absence of dystrophin, and oligomer therapy may be able to normalise metabolic activity and restore contractility in mdx mouse hearts