Emerging methods for and novel insights gained by absolute quantification of mitochondrial DNA copy number and its clinical applications.

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.

Using hiPSC-CMs to Examine Mechanisms of Catecholaminergic Polymorphic Ventricular Tachycardia.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a potentially lethal inherited cardiac arrhythmia condition, triggered by physical or acute emotional stress, that predominantly expresses early in life. Gain-of-function mutations in the cardiac ryanodine receptor gene (RYR2) account for the majority of CPVT cases, causing substantial disruption of intracellular calcium (Ca2+ ) homeostasis particularly during the periods of ß-adrenergic receptor stimulation. However, the highly variable penetrance, patient outcomes, and drug responses observed in clinical practice remain unexplained, even for patients with well-established founder RyR2 mutations. Therefore, investigation of the electrophysiological consequences of CPVT-causing RyR2 mutations is crucial to better understand the pathophysiology of the disease. The development of strategies for reprogramming human somatic cells to human induced pluripotent stem cells (hiPSCs) has provided a unique opportunity to study inherited arrhythmias, due to the ability of hiPSCs to differentiate down a cardiac lineage. Employment of genome editing enables generation of disease-specific cell lines from healthy and diseased patient-derived hiPSCs, which subsequently can be differentiated into cardiomyocytes. This paper describes the means for establishing an hiPSC-based model of CPVT in order to recapitulate the disease phenotype in vitro and investigate underlying pathophysiological mechanisms. The framework of this approach has the potential to contribute to disease modeling and personalized medicine using hiPSC-derived cardiomyocytes. © 2021 Wiley Periodicals LLC.

A differentiated Ca2+ signalling phenotype has minimal impact on myocardin expression in an automated differentiation assay using A7r5 cells.

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 Ca2+ signalling in electrically coupled, differentiated cells to increased dependence on store-operated Ca2+ entry and sarcoplasmic reticulum Ca2+ release in synthetic cells. Conversely, an increased voltage-gated Ca2+ entry is seen when proliferating A7r5 smooth muscle cells quiesce. We asked whether this change in Ca2+ signalling was linked to changes in the expression of the phenotype-regulating transcriptional co-activator myocardin or α-smooth muscle actin, using correlative epifluorescence Ca2+ 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 Ca2+ signalling showed increasing dependence on L-type voltage-gated Ca2+ 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 Ca2+ 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 Ca2+ signalling phenotype, while chronic inhibition of voltage-gated Ca2+ entry modestly increased immunoreactivity of myocardin. Stepwise regression analyses suggested that changes in myocardin expression had a weak relationship with Ca2+ signalling synchronicity, but not frequency or amplitude. In conclusion, we report a 96-well assay and analytical pipeline to study the link between Ca2+ 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 Ca2+ signalling phenotype.

Mechanisms of Arrhythmogenicity of Hypertrophic Cardiomyopathy-Associated Troponin T (TNNT2) Variant I79N.

Hypertrophic cardiomyopathy (HCM) is the most common heritable cardiovascular disease and often results in cardiac remodeling and an increased incidence of sudden cardiac arrest (SCA) and death, especially in youth and young adults. Among thousands of different variants found in HCM patients, variants of TNNT2 (cardiac troponin T-TNNT2) are linked to increased risk of ventricular arrhythmogenesis and sudden death despite causing little to no cardiac hypertrophy. Therefore, studying the effect of TNNT2 variants on cardiac propensity for arrhythmogenesis can pave the way for characterizing HCM in susceptible patients before sudden cardiac arrest occurs. In this study, a TNNT2 variant, I79N, was generated in human cardiac recombinant/reconstituted thin filaments (hcRTF) to investigate the effect of the mutation on myofilament Ca2+ sensitivity and Ca2+ dissociation rate using steady-state and stopped-flow fluorescence techniques. The results revealed that the I79N variant significantly increases myofilament Ca2+ sensitivity and decreases the Ca2+ off-rate constant (k off). To investigate further, a heterozygous I79N+/- TNNT2 variant was introduced into human-induced pluripotent stem cells using CRISPR/Cas9 and subsequently differentiated into ventricular cardiomyocytes (hiPSC-CMs). To study the arrhythmogenic properties, monolayers of I79N+/- hiPSC-CMs were studied in comparison to their isogenic controls. Arrhythmogenesis was investigated by measuring voltage (V m) and cytosolic Ca2+ transients over a range of stimulation frequencies. An increasing stimulation frequency was applied to the cells, from 55 to 75 bpm. The results of this protocol showed that the TnT-I79N cells had reduced intracellular Ca2+ transients due to the enhanced cytosolic Ca2+ buffering. These changes in Ca2+ handling resulted in beat-to-beat instability and triangulation of the cardiac action potential, which are predictors of arrhythmia risk. While wild-type (WT) hiPSC-CMs were accurately entrained to frequencies of at least 150 bpm, the I79N hiPSC-CMs demonstrated clear patterns of alternans for both V m and Ca2+ transients at frequencies >75 bpm. Lastly, a transcriptomic analysis was conducted on WT vs. I79N+/- TNNT2 hiPSC-CMs using a custom NanoString codeset. The results showed a significant upregulation of NPPA (atrial natriuretic peptide), NPPB (brain natriuretic peptide), Notch signaling pathway components, and other extracellular matrix (ECM) remodeling components in I79N+/- vs. the isogenic control. This significant shift demonstrates that this missense in the TNNT2 transcript likely causes a biophysical trigger, which initiates this significant alteration in the transcriptome. This TnT-I79N hiPSC-CM model not only reproduces key cellular features of HCM-linked mutations but also suggests that this variant causes uncharted pro-arrhythmic changes to the human action potential and gene expression.

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

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 Ca2+ influx through specific voltage-gated Ca2+ 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.

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

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.