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Localization of sodium channel subtypes in mouse ventricular myocytes using quantitative immunocytochemistry.

ABSTRACT: Voltage-gated sodium channels are responsible for the rising phase of the action potential in cardiac muscle. Previously, both TTX-sensitive neuronal sodium channels (NaV1.1, NaV1.2, NaV1.3, NaV1.4 and NaV1.6) and the TTX-resistant cardiac sodium channel (NaV1.5) have been detected in cardiac myocytes, but relative levels of protein expression of the isoforms were not determined. Using a quantitative approach, we analyzed z-series of confocal microscopy images from individual mouse myocytes stained with either anti-NaV1.1, anti-NaV1.2, anti-NaV1.3, anti-NaV1.4, anti-NaV1.5, or anti-NaV1.6 antibodies and calculated the relative intensity of staining for these sodium channel isoforms. Our results indicate that the TTX-sensitive channels represented approximately 23% of the total channels, whereas the TTX-resistant NaV1.5 channel represented 77% of the total channel staining in mouse ventricular myocytes. These ratios are consistent with previous electrophysiological studies in mouse ventricular myocytes. NaV1.5 was located at the cell surface, with high density at the intercalated disc, but was absent from the transverse (t)-tubular system, suggesting that these channels support surface conduction and inter-myocyte transmission. Low-level cell surface staining of NaV1.4 and NaV1.6 channels suggest a minor role in surface excitation and conduction. Conversely, NaV1.1 and NaV1.3 channels are localized to the t-tubules and are likely to support t-tubular transmission of the action potential to the myocyte interior. This quantitative immunocytochemical approach for assessing sodium channel density and localization provides a more precise view of the relative importance and possible roles of these individual sodium channel protein isoforms in mouse ventricular myocytes and may be applicable to other species and cardiac tissue types.

SUBMITTER: Westenbroek RE

PROVIDER: S-EPMC3851329 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Localization of sodium channel subtypes in mouse ventricular myocytes using quantitative immunocytochemistry.

Westenbroek Ruth E RE Bischoff Sebastian S Fu Ying Y Maier Sebastian K G SK Catterall William A WA Scheuer Todd T

Journal of molecular and cellular cardiology 20130824

Voltage-gated sodium channels are responsible for the rising phase of the action potential in cardiac muscle. Previously, both TTX-sensitive neuronal sodium channels (NaV1.1, NaV1.2, NaV1.3, NaV1.4 and NaV1.6) and the TTX-resistant cardiac sodium channel (NaV1.5) have been detected in cardiac myocytes, but relative levels of protein expression of the isoforms were not determined. Using a quantitative approach, we analyzed z-series of confocal microscopy images from individual mouse myocytes stai ...[more]

PMID: 23982034

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Project description:Transient receptor potential canonical 1 (TRPC1) protein is abundantly expressed in cardiomyocytes. While TRPC1 is supposed to be critically involved in cardiac hypertrophy, its physiological role in cardiomyocytes is poorly understood. We investigated the subcellular location of TRPC1 and its contribution to Ca2+ signaling in mammalian ventricular myocytes. Immunolabeling, three-dimensional scanning confocal microscopy and quantitative colocalization analysis revealed an abundant intracellular location of TRPC1 in neonatal rat ventricular myocytes (NRVMs) and adult rabbit ventricular myocytes. TRPC1 was colocalized with intracellular proteins including sarco/endoplasmic reticulum Ca2+ ATPase 2 in the sarcoplasmic reticulum (SR). Colocalization with wheat germ agglutinin, which labels the glycocalyx and thus marks the sarcolemma including the transverse tubular system, was low. Super-resolution and immunoelectron microscopy supported the intracellular location of TRPC1. We investigated Ca2+ signaling in NRVMs after adenoviral TRPC1 overexpression or silencing. In NRVMs bathed in Na+ and Ca2+ free solution, TRPC1 overexpression and silencing was associated with a decreased and increased SR Ca2+ content, respectively. In isolated rabbit cardiomyocytes bathed in Na+ and Ca2+ free solution, we found an increased decay of the cytosolic Ca2+ concentration [Ca2+]i and increased SR Ca2+ content in the presence of the TRPC channel blocker SKF-96365. In a computational model of rabbit ventricular myocytes at physiological pacing rates, Ca2+ leak through SR TRPC channels increased the systolic and diastolic [Ca2+]i with only minor effects on the action potential and SR Ca2+ content. Our studies suggest that TRPC1 channels are localized in the SR, and not present in the sarcolemma of ventricular myocytes. The studies provide evidence for a role of TRPC1 as a contributor to SR Ca2+ leak in cardiomyocytes, which was previously explained by ryanodine receptors only. We propose that the findings will guide us to an understanding of TRPC1 channels as modulators of [Ca2+]i and contractility in cardiomyocytes.

Project description:AimsRecent studies have demonstrated electrotonic coupling between scar tissue and the surrounding myocardium in cryoinjured hearts. However, the electrical dynamics occurring at the myocyte-nonmyocyte interface in the fibrotic heart remain undefined. Here, we sought to develop an assay to interrogate the nonmyocyte cell type contributing to heterocellular coupling and to characterize, on a cellular scale, its voltage response in the infarct border zone of living hearts.Methods and resultsWe used two-photon laser scanning microscopy in conjunction with a voltage-sensitive dye to record transmembrane voltage changes simultaneously from cardiomyocytes and adjoined nonmyocytes in Langendorff-perfused mouse hearts with healing myocardial infarction. Transgenic mice with cardiomyocyte-restricted expression of a green fluorescent reporter protein underwent permanent coronary artery ligation and their hearts were subjected to voltage imaging 7-10 days later. Reporter-negative cells, i.e. nonmyocytes, in the infarct border zone exhibited depolarizing transients at a 1:1 coupling ratio with action potentials recorded simultaneously from adjacent, reporter-positive ventricular myocytes. The electrotonic responses in the nonmyocytes exhibited slower rates of de- and repolarization compared to the action potential waveform of juxtaposed myocytes. Voltage imaging in infarcted hearts expressing a fluorescent reporter specifically in myofibroblasts revealed that the latter were electrically coupled to border zone myocytes. Their voltage transient properties were indistinguishable from those of nonmyocytes in hearts with cardiomyocyte-restricted reporter expression. The density of connexin43 expression at myofibroblast-cardiomyocyte junctions was ∼5% of that in the intercalated disc regions of paired ventricular myocytes in the remote, uninjured myocardium, whereas the ratio of connexin45 to connexin43 expression levels at heterocellular contacts was ∼1%.ConclusionMyofibroblasts contribute to the population of electrically coupled nonmyocytes in the infarct border zone. The slower kinetics of myofibroblast voltage responses may reflect low electrical conductivity across heterocellular junctions, in accordance with the paucity of connexin expression at myofibroblast-cardiomyocyte contacts.

Dataset Information

Localization of sodium channel subtypes in mouse ventricular myocytes using quantitative immunocytochemistry.

Publications

Localization of sodium channel subtypes in mouse ventricular myocytes using quantitative immunocytochemistry.

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