Ion channel mRNA distribution and expression in the sinoatrial node and right atrium of dogs and monkeys

Journal of Toxicologic Pathology

2021 Apr 17

SANO, T;YASUNO, H;WATANABE, T;
| DOI: 10.1293/tox.2020-0089

There are limited data on the gene expression profiles of ion channels in the sinoatrial node (SAN) of dogs and monkeys. In this study, the messenger RNA (mRNA) expression profiles of various ion channels in the SAN of naïve dogs and monkeys were examined using RNAscope _in situ _hybridization and compared with those in the surrounding right atrium (RA) of each species. Regional-specific Cav1.3 and HCN4 expression was observed in the SAN of dogs and monkeys. Additionally, HCN1 in dogs was only expressed in the SAN group. The expression profiles of Cav3.1 and Cav3.2 in the SAN and RA were completely different between dogs and monkeys. Dog hearts only expressed Cav3.2; however, Cav3.1 was detected only in monkeys, and the expression score in the SAN was slightly higher than that in the RA. Although Kir3.1 and NCX1 in dogs were equally expressed in both the SAN and RA, the expression scores of these genes in the SAN of monkeys were slightly higher than those in the RA. The Kir3.4 expression score in the SAN of dogs and monkeys was also slightly higher than that in the RA. The mRNA expression scores of Kv11.1/ERG and KvLQT1 were equally observed in both the SAN and RA of dogs and monkeys. HCN2 was not detected in dogs and monkeys. In summary, we used RNAscope to demonstrate the SAN-specific gene expression patterns of ion channels, which may be useful in explaining the effect of pacemaking and/or hemodynamic effects in nonclinical studies.

Heart failure (HF) alters distribution and spatial association profiles of calmodulin (CaM) and CaM target protein mRNAs

Biophysical Journal

2023 Feb 01

Bogdanov, V;Soltisz, A;Beard, C;Hernandez Orengo, B;Sakuta, G;Veeraraghavan, R;Davis, J;Gyorke, S;
| DOI: 10.1016/j.bpj.2022.11.1389

Aberrant Ca-CaM signaling has been implicated in various congenital and acquired cardiac pathologies, including arrhythmia, hypertrophy, and HF. We examined the impact of HF induced by trans-aortic constriction (TAC) on the distribution of the three CaM mRNAs (Calm 1,2 and 3) and their key protein target mRNAs (Ryr2, Scn5a, Camk2d, NOS1 and Cacna1c) in cardiomyocytes, using fluorescence in situ hybridization (RNAScope™). HF resulted in specific changes in the pattern of localization of Calms, manifested in redistribution of Calm3 from the cell periphery towards the perinuclear area and enhanced Calm2 attraction to the perinuclear area compared to sham myocytes. Additionally, HF resulted in redistribution of mRNAs for certain CaM target mRNAs. Particularly, NOS1 localization shifted from the cell periphery towards the perinuclear area, Cacna1c, Camk2d and Scn5a abundance increased at the perinuclear area, and Ryr2 attracted even closer to the cell periphery in HF myocytes compared to sham myocytes. The strength of non-random attraction/repulsion was measured as the maximal deviation between the observed distribution of nearest neighbor distances from the distribution predicted under complete spatial randomness. Consistent with the observed alterations in abundance and distribution of CaM and CaM target mRNAs, HF resulted in increased attraction between Calm1 and Scn5a, Ryr2 and Camk2d, between Calm2 and Ryr2 and Camk2d; and between Calm3 and NOS1 and Scn5a. In contrast, the attraction between Calm3 and Ryr2 decreased in HF myocytes compared to sham. Collectively, these results suggest distribution of Calms and their association with key target protein mRNAs undergo substantial alterations in heart failure. These results have new important implications for organization of Ca signaling in normal and diseased heart.

Genetic silencing of striatal CaV1.3 prevents and ameliorates levodopa dyskinesia.

Mov Disord.

2019 Apr 19

Steece-Collier K, Stancati JA, Collier NJ, Sandoval IM, Mercado NM, Sortwell CE, Collier TJ, Manfredsson FP.
PMID: 31002755 | DOI: 10.1002/mds.27695

Abstract

BACKGROUND:

Levodopa-induced dyskinesias are an often debilitating side effect of levodopa therapy in Parkinson's disease. Although up to 90% of individuals with PD develop this side effect, uniformly effective and well-tolerated antidyskinetic treatment remains a significant unmet need. The pathognomonic loss of striatal dopamine in PD results in dysregulation and disinhibition of striatal CaV1.3 calcium channels, leading to synaptopathology that appears to be involved in levodopa-induced dyskinesias. Although there are clinically available drugs that can inhibit CaV1.3 channels, they are not adequately potent and have only partial and transient impact on levodopa-induced dyskinesias.

METHODS:

To provide unequivocal target validation, free of pharmacological limitations, we developed a CaV1.3 shRNA to provide high-potency, target-selective, mRNA-level silencing of striatal CaV1.3 channels and examined its ability to impact levodopa-induced dyskinesias in severely parkinsonian rats.

RESULTS:

We demonstrate that vector-mediated silencing of striatal CaV1.3 expression in severely parkinsonian rats prior to the introduction of levodopa can uniformly and completely prevent induction of levodopa-induced dyskinesias, and this antidyskinetic benefit persists long term and with high-dose levodopa. In addition, this approach is capable of ameliorating preexisting severe levodopa-induced dyskinesias. Importantly, motoric responses to low-dose levodopa remained intact in the presence of striatal CaV1.3 silencing, indicating preservation of levodopa benefit without dyskinesia liability.

DISCUSSION:

The current data provide some of the most profound antidyskinetic benefit reported to date and suggest that genetic silencing of striatal CaV1.3 channels has the potential to transform treatment of individuals with PD by allowing maintenance of motor benefit of levodopa in the absence of the debilitating levodopa-induced dyskinesia side effect.

Caveolae-restricted mechano-chemical signal transduction in mouse atrial myocytes

Biophysical Journal

2023 Feb 01

Medvedev, R;Turner, D;Gorelik, J;Alvarado, F;Bondarenko, V;Glukhov, A;
| DOI: 10.1016/j.bpj.2022.11.1392

Atrial fibrillation (AF) is commonly observed in patients with hypertension and is associated with pathologically elevated cardiomyocyte stretch. AF triggers have been linked to subcellular Ca2+ abnormalities, while their association with stretch remains elusive. Caveolae are mechanosensitive membrane structures, that play a role in both Ca2+ and cyclic adenosine monophosphate (cAMP) signaling. Therefore, caveolae could provide a mechanistic connection between cardiomyocyte stretch, Ca2+ mishandling, and AF. In isolated mouse atrial myocytes, cell stretch was mimicked by hypotonic swelling, which increased cell width (by ∼30%, p

An integrate-and-fire approach to Ca2+ signaling—The noise of puffs

Biophysical Journal

2023 Feb 01

Ramlow, L;Falcke, M;Lindner, B;
| DOI: 10.1016/j.bpj.2022.11.1390

Stochastic spiking is a prominent feature of Ca2+ signaling. The main noise source at the cellular level are puffs from inositol-trisphosphate receptor (IP3R) channel clusters in the membrane of the endoplasmic reticulum (ER). While the random cluster activity has been known for decades, a stringent method to derive the puff noise term acting on the cytosolic Ca2+ concentration is still lacking. We adopt a popular description of neural spike generation from neuroscience, the stochastic integrate-and-fire (IF) model, to describe Ca2+ spiking. Our model consists of two components describing i) activity of IP3R clusters and ii) dynamics of the global Ca2+ concentrations in the cytosol and in the ER. Cluster activity is modeled by a Markov chain, capturing the puff. The global Ca2+ concentrations are described by a two-variable IF model driven by the puff current. For the Markov chain we derive expressions for the statistics of interpuff interval, single-puff strength, and puff current assuming constant cytosolic Ca2+, an assumption often well met because the Ca2+ concentrations vary much slower than the cluster activity does. The latter assumption also allows to approximate the driving Ca2+ dependent puff current by a white Gaussian noise. This approximation results in an IF model with nonlinear drift and multiplicative noise. We consider this reduced model in a renewal version and in a version with cumulative refractoriness. Neglecting ER depletion, the stochastic IF model has only one variable and generates a renewal spike train, a point process with statistically independent interspike intervals (ISI). We derive analytical expressions for the mean and coefficient of variation of the ISI and suggest approximations for the ISI density and spike-train power spectrum. Taking into account ER depletion, the two-variable IF model displays cumulative refractoriness as seen in experimental data.

Physiological significance of tissue-specific MICU regulation of mitochondrial calcium uptake

Biophysical Journal

2023 Feb 01

Rodriguez, M;Tsai, C;Tsai, M;
| DOI: 10.1016/j.bpj.2022.11.1391

The mitochondrial calcium uniporter is a multi-subunit calcium channel that imports Ca2+ into mitochondria. Its MICU subunits (MICU1, MICU2, and the neuron-specific MICU3) gate the channel by blocking the pore in low Ca2+. Upon local Ca2+ elevation, Ca2+ binds to MICUs to cause MICU unblock, thus opening the pore so Ca2+ can permeate. Previous work using cell lines suggests that the uniporter in mammalian cells is exclusively regulated by a MICU1-MICU2 heterodimer. However, we show here that multiple types of electrically excitable cells, including skeletal muscle and cardiac tissues, can also possess a MICU1-MICU1 homodimer or virtually no MICUs. Kinetic analyses demonstrate that MICU1 has a higher Ca2+ affinity than MICU2, and that without MICUs the uniporter is constitutively open. As a result, uniporters with the MICU1-1 homodimer or no MICUs exhibit higher transport activities, leading to mitochondria accumulating much higher levels of matrix Ca2+. Using a Seahorse assay, we show that cells with MICU1-1 or no MICUs have impaired basal oxidative phosphorylation, likely due to increased ROS and damaged respiratory-complex proteins, including NDUFS3 and COX2. These cells, moreover, are highly susceptible to apoptosis. The disadvantage of employing MICU1-1 or omitting MICUs, however, accompanies strong physiological benefits. We show that in response to intracellular Ca2+ signals, these mitochondria import more Ca2+ and consequently produce more ATP, thus better supplying the energy required for the cellular processes initiated by the Ca2+ signals. In conclusion, this work reveals that tissues can manipulate their mitochondrial calcium uptake properties to suit their unique physiological needs by customizing their MICU regulation of the mitochondrial calcium uniporter.

	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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