crispr-cas9-mediated-precise-knock-in-edits-in-zebrafish-hearts

CRISPR-Cas9-Mediated Precise Knock-In Edits in Zebrafish Hearts

Clustered regularly interspaced short palindromic repeats (CRISPR) in animal models enable precise genetic manipulation for the study of physiological ...

Simon Fraser University

Mitochondria convert the energy stored in carbohydrate and fat into ATP molecules that power enzymatic reactions within cells, and this process influences cellular calcium signals in several ways. By providing ATP to calcium pumps at the plasma and intracellular membranes, mitochondria power the calcium gradients that drive the release of Ca²⁺ from stores and the entry of Ca²⁺ across plasma membrane channels. By taking up and subsequently releasing calcium ions, mitochondria determine the spatiotemporal profile of cellular Ca²⁺ signals and the activity of Ca²⁺-regulated proteins, including Ca²⁺ entry channels that are themselves part of the Ca²⁺ circuitry. Ca²⁺ elevations in the mitochondrial matrix, in turn, activate Ca²⁺-dependent enzymes that boost the respiratory chain, increasing the ability of mitochondria to buffer calcium ions. Mitochondria are able to encode and decode Ca²⁺ signals because the respiratory chain generates an electrochemical gradient for protons across the inner mitochondrial membrane. This proton motive force (Δp) drives the activity of the ATP synthase and has both an electrical component, the mitochondrial membrane potential (ΔΨ(m)), and a chemical component, the mitochondrial proton gradient (ΔpH(m)). ΔΨ(m) contributes about 190 mV to Δp and drives the entry of Ca²⁺ across a recently identified Ca²⁺-selective channel known as the mitochondrial Ca²⁺ uniporter. ΔpH(m) contributes ~30 mV to Δp and is usually ignored or considered a minor component of mitochondria respiratory state. However, the mitochondrial proton gradient is an essential component of the chemiosmotic theory formulated by Peter Mitchell in 1961 as ΔpH(m) sustains the entry of substrates and metabolites required for the activity of the respiratory chain and drives the activity of electroneutral ion exchangers that allow mitochondria to maintain their osmolarity and volume. In this review, we summarize the mechanisms that regulate the mitochondrial proton gradient and discuss how thermodynamic concepts derived from measurements in purified mitochondria can be reconciled with our recent findings that mitochondria have high proton permeability in situ and that ΔpH(m) decreases during mitochondrial Ca²⁺ elevations.

Regulation of the mitochondrial proton gradient by cytosolic Ca²⁺ signals.

The chemical nature and functional significance of mitochondrial flashes associated with fluctuations in mitochondrial membrane potential is unclear. Using a ratiometric pH probe insensitive to superoxide, we show that flashes reflect matrix alkalinization transients of ∼0.4 pH units that persist in cells permeabilized in ion-free solutions and can be evoked by imposed mitochondrial depolarization. Ablation of the pro-fusion protein Optic atrophy 1 specifically abrogated pH flashes and reduced the propagation of matrix photoactivated GFP (paGFP). Ablation or invalidation of the pro-fission Dynamin-related protein 1 greatly enhanced flash propagation between contiguous mitochondria but marginally increased paGFP matrix diffusion, indicating that flashes propagate without matrix content exchange. The pH flashes were associated with synchronous depolarization and hyperpolarization events that promoted the membrane potential equilibration of juxtaposed mitochondria. We propose that flashes are energy conservation events triggered by the opening of a fusion pore between two contiguous mitochondria of different membrane potentials, propagating without matrix fusion to equilibrate the energetic state of connected mitochondria. 

OPA1 promotes pH flashes that spread between contiguous mitochondria without matrix protein exchange.

KCNQ5 is a highly conserved gene encoding an important channel for neuronal function; it is widely expressed in the brain and generates M-type current. Exome sequencing identified de novo heterozygous missense mutations in four probands with intellectual disability, abnormal neurological findings, and treatment-resistant epilepsy (in two of four). Comprehensive analysis of this potassium channel for the four variants expressed in frog oocytes revealed shifts in the voltage dependence of activation, including altered activation and deactivation kinetics. Specifically, both loss-of-function and gain-of-function KCNQ5 mutations, associated with increased excitability and decreased repolarization reserve, lead to pathophysiology.

Loss-of-Function and Gain-of-Function Mutations in KCNQ5 Cause Intellectual Disability or Epileptic Encephalopathy.

Absolute quantification of mitochondrial DNA copy number (mCN) provides important insights in many fields of research including cancer, cardiovascular and reproductive health. Droplet digital PCR (ddPCR) natively reports absolute copy number, and we have developed a single-dye, multiplex assay to measure rat mCN that is accurate, precise and affordable. We demonstrate simple methods to optimize this assay and to determine nuclear reference pseudogene copy number to extend the range of mCN that can be measured with this assay. We evaluated two commonly used mitochondrial DNA reference loci to determine mCN, the ND1 gene and the D-Loop. Harnessing the absolute measures of ddPCR, we found that the D-Loop amplifies with a copy number of ~1.0-1.5 relative to other sites on the mitochondrial genome. This anomalous copy number varied significantly between rats and tissues (aorta, brain, heart, liver, soleus muscle). We advocate for avoiding the D-Loop as a mitochondrial reference in future studies of mCN. Further, we report a novel approach to quantifying immunolabelled mitochondrial DNA that provides single-cell estimates of mCN that closely agree with the population analyses by ddPCR. The combination of these assays represents a cost-effective and powerful suite of tools to study mCN.

Droplet digital PCR shows the D-Loop to be an error prone locus for mitochondrial DNA copy number determination.

ATP and norepinephrine (NE) are coreleased from peripheral sympathetic nerve terminals. Whether they are stored in the same vesicles has been debated for decades. Preferential dependence of NE or ATP release on Ca influx through specific voltage-gated Ca channel (Cav2) isoforms suggests that NE and ATP are stored in separate vesicle pools, but simultaneous imaging of NE and ATP containing vesicles within single varicosities has not been reported. We conducted an immunohistochemical study of vesicular monoamine transporter 2 (VMAT2/SLC18A2) and vesicular nucleotide translocase (VNUT/SLC17A9) as markers of vesicles containing NE and ATP in sympathetic nerves of the rat tail artery. A large fraction of varicosities exhibited neighboring, rather than overlapping, VNUT and VMAT2 fluorescent puncta. VMAT2, but not VNUT, colocalized with synaptotagmin 1. Cav2.1, Cav2.2, and Cav2.3 are expressed in nerves in the tunica adventitia. VMAT2 preferentially localized adjacent to Cav2.2 and Cav2.3 rather than Cav2.1. VNUT preferentially localized adjacent to Cav2.3 > Cav2.2 >> Cav2.1. With the use of wire myography, inhibition of field-stimulated vasoconstriction with the Cav2.3 blocker SNX-482 (0.25 µM) mimicked the effects of the P2X inhibitor suramin (100 µM) rather than the α-adrenergic inhibitor phentolamine (10 µM). Variable sensitivity to SNX-482 and suramin between animals closely correlated with Cav2.3 staining. We concluded that a majority of ATP and NE stores localize to separate vesicle pools that use different synaptotagmin isoforms and that localize near different Cav2 isoforms to mediate vesicle release. Cav2.3 appears to play a previously unrecognized role in mediating ATP release in the rat tail artery. NEW & NOTEWORTHY Immunofluorescence imaging of vesicular nucleotide translocase and vesicular monoamine transporter 2 in rat tail arteries revealed that ATP and norepinephrine, classical cotransmitters, localize to well-segregated vesicle pools. Furthermore, vesicular nucleotide translocase and vesicular monoamine transporter 2 exhibit preferential localization with specific Cav2 isoforms. These novel observations address long-standing debates regarding the mechanism(s) of sympathetic neurotransmitter corelease.

VNUT and VMAT2 segregate within sympathetic varicosities and localize near preferred Cav2 isoforms in the rat tail artery.

Dravet syndrome is a severe form of childhood epilepsy characterized by frequent temperature-sensitive seizures and delays in cognitive development. In the majority (80%) of cases, Dravet syndrome is caused by mutations in the SCN1A gene, encoding the voltage-gated sodium channel Na1.1, which is abundant in the central nervous system. Dravet syndrome can be caused by either gain-of-function mutation or loss of function in Na1.1, making it necessary to characterize each novel mutation. Here we use a combination of patch-clamp recordings and immunocytochemistry to characterize the first known NH-terminal amino acid duplication mutation found in a patient with Dravet syndrome, M72dup. M72dup does not significantly alter rate of fast inactivation recovery or rate of fast inactivation onset at any measured membrane potential. M72dup significantly shifts the midpoint of the conductance voltage relationship to more hyperpolarized potentials. Most interestingly, M72dup significantly reduces peak current of Na1.1 and reduces membrane expression. This suggests that M72dup acts as a loss-of-function mutation primarily by impacting the ability of the channel to localize to the plasma membrane. Genetic screening of a patient with Dravet syndrome revealed a novel mutation in SCN1A. Of over 700 SCN1A mutations known to cause Dravet syndrome, M72dup is the first to be identified in the NH-terminus of Na1.1. We studied M72dup using patch-clamp electrophysiology and immunocytochemistry. M72dup causes a decrease in membrane expression of Na1.1 and overall loss of function, consistent with the role of the NH-terminal region in membrane trafficking of Na1.1.

Case studies in neuroscience: a novel amino acid duplication in the NH-terminus of the brain sodium channel Na1.1 underlying Dravet syndrome.

Animal studies suggest that decreased vascular mitochondrial DNA copy number can promote hypertension. We conducted a chart review of blood pressure and hemodynamics in patients with either mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS, n = 36) or individuals with variants in the mitochondrial DNA polymerase gamma (POLG, n = 26). The latter included both pathogenic variants and variants of unknown significance (VUS). Hypertension rates (MELAS 50%, POLG 50%) were elevated relative to Canadian norms in 20-39 (MELAS) and 40-59 (MELAS and POLG) years of age groups. Peripheral resistance was high in the hypertensive versus normotensive patients, potentially indicative of microvascular disease. Despite antihypertensive treatment, systolic blood pressure remained elevated in the POLG versus MELAS group. The risk of hypertension was not associated with MELAS heteroplasmy. Hypertension rates were not different between individuals with known pathogenic POLG variants and those with VUS, including common variants. Hypertension (HT) also did not differ between patients with POLG variants with (n = 17) and without chronic progressive external opthalmoplegia (n = 9) (CPEO). HT was associated with variants in all three functional domains of POLG. These findings suggest that both pathogenic variants and several VUS in the POLG gene may promote human hypertension and extend our past reports that increased risk of HT is associated with MELAS.

High rate of hypertension in patients with m.3243A>G MELAS mutations and POLG variants.

Vascular smooth muscle cells are unusual in that differentiated, contractile cells possess the capacity to "de-differentiate" into a synthetic phenotype that is characterized by being replicative, secretory, and migratory. One aspect of this phenotypic modulation is a shift from voltage-gated Ca signalling in electrically coupled, differentiated cells to increased dependence on store-operated Ca entry and sarcoplasmic reticulum Ca release in synthetic cells. Conversely, an increased voltage-gated Ca entry is seen when proliferating A7r5 smooth muscle cells quiesce. We asked whether this change in Ca signalling was linked to changes in the expression of the phenotype-regulating transcriptional co-activator myocardin or α-smooth muscle actin, using correlative epifluorescence Ca imaging and immunocytochemistry. Cells were cultured in growth media (DMEM, 10% serum, 25 mM glucose) or differentiation media (DMEM, 1% serum, 5 mM glucose). Coinciding with growth arrest, A7r5 cells became electrically coupled, and spontaneous Ca signalling showed increasing dependence on L-type voltage-gated Ca channels that were blocked with nifedipine (5 μM). These synchronized oscillations were modulated by ryanodine receptors, based on their sensitivity to dantrolene (5 μM). Actively growing cultures had spontaneous Ca transients that were insensitive to nifedipine and dantrolene but were blocked by inhibition of the sarco-endoplasmic reticulum ATPase with cyclopiazonic acid (10 μM). In cells treated with differentiation media, myocardin and αSMA immunoreactivity increased prior to changes in the Ca signalling phenotype, while chronic inhibition of voltage-gated Ca entry modestly increased immunoreactivity of myocardin. Stepwise regression analyses suggested that changes in myocardin expression had a weak relationship with Ca signalling synchronicity, but not frequency or amplitude. In conclusion, we report a 96-well assay and analytical pipeline to study the link between Ca signalling and smooth muscle differentiation. This assay showed that changes in the expression of two molecular differentiation markers (myocardin and αSMA) tended to precede changes in the Ca signalling phenotype.

A differentiated Ca signalling phenotype has minimal impact on myocardin expression in an automated differentiation assay using A7r5 cells.

Cannabidiol (CBD) is the primary nonpsychotropic phytocannabinoid found in Cannabis sativa, which has been proposed to be therapeutic against many conditions, including muscle spasms. Among its putative targets are voltage-gated sodium channels (Navs), which have been implicated in many conditions. We investigated the effects of CBD on Nav1.4, the skeletal muscle Nav subtype. We explored direct effects, involving physical block of the Nav pore, as well as indirect effects, involving modulation of membrane elasticity that contributes to Nav inhibition. MD simulations revealed CBD's localization inside the membrane and effects on bilayer properties. Nuclear magnetic resonance (NMR) confirmed these results, showing CBD localizing below membrane headgroups. To determine the functional implications of these findings, we used a gramicidin-based fluorescence assay to show that CBD alters membrane elasticity or thickness, which could alter Nav function through bilayer-mediated regulation. Site-directed mutagenesis in the vicinity of the Nav1.4 pore revealed that removing the local anesthetic binding site with F1586A reduces the block of INa by CBD. Altering the fenestrations in the bilayer-spanning domain with Nav1.4-WWWW blocked CBD access from the membrane into the Nav1.4 pore (as judged by MD). The stabilization of inactivation, however, persisted in WWWW, which we ascribe to CBD-induced changes in membrane elasticity. To investigate the potential therapeutic value of CBD against Nav1.4 channelopathies, we used a pathogenic Nav1.4 variant, P1158S, which causes myotonia and periodic paralysis. CBD reduces excitability in both wild-type and the P1158S variant. Our in vitro and in silico results suggest that CBD may have therapeutic value against Nav1.4 hyperexcitability.

Cannabidiol inhibits the skeletal muscle Nav1.4 by blocking its pore and by altering membrane elasticity.

The past thirty years have seen a surge in interest in pathophysiological roles of mitochondria, and the accurate quantification of mitochondrial DNA copy number (mCN) in cells and tissue samples is a fundamental aspect of assessing changes in mitochondrial health and biogenesis. Quantification of mCN between studies is surprisingly variable due to a combination of physiological variability and diverse protocols being used to measure this endpoint. The advent of novel methods to quantify nucleic acids like digital polymerase chain reaction (dPCR) and high throughput sequencing offer the ability to measure absolute values of mCN. We conducted an in-depth survey of articles published between 1969 -- 2020 to create an overview of mCN values, to assess consensus values of tissue-specific mCN, and to evaluate consistency between methods of assessing mCN. We identify best practices for methods used to assess mCN, and we address the impact of using specific loci on the mitochondrial genome to determine mCN. Current data suggest that clinical measurement of mCN can provide diagnostic and prognostic value in a range of diseases and health conditions, with emphasis on cancer and cardiovascular disease, and the advent of means to measure absolute mCN should improve future clinical applications of mCN measurements.

Damon Poburko

Department of Biomedical Physiology and Kinesiology

Simon Fraser University

CRISPR-Cas9-Mediated Precise Knock-In Edits in Zebrafish Hearts