Nuclear Ca(2+) regulates key cellular processes, including gene expression, apoptosis, assembly of the nuclear envelope, and nucleocytoplasmic transport. Quantification of subcellularly resolved Ca(2+) signals is, therefore, essential for understanding physiological and pathological processes in various cell types. However, the properties of commonly used Ca(2+)-fluorescent indicators in intracellular compartments may differ, thus affecting the translation of Ca(2+)-dependent fluorescence changes into quantitative changes of Ca(2+) concentration. Here, we describe technical approaches for reliable subcellular quantification of [Ca(2+)] in the cytoplasm vs. the nucleus and the nuclear envelope by in situ calibration of fluorescein-derived fluorescent indicators Fluo-4 and Fluo-5N.

Calcium (Ca) is a universal second messenger involved in the regulation of various cellular processes, including electrical signaling, contraction, secretion, memory, gene transcription, and cell death. In heart, Ca governs cardiomyocyte contraction, is central in electrophysiological properties, and controls major signaling pathway implicated in gene transcription. How cardiomyocytes decode Ca signal to regulate gene expression without interfering with, or being controlled by, "contractile" Ca that floods the entire cytosol during each heartbeat is still elusive. In this review, we summarize recent findings on nuclear Ca regulation and its downstream signaling in cardiomyocytes. We will address difficulties in reliable quantification of nuclear Ca fluxes and discuss its role in the development and progression of cardiac hypertrophy and heart failure. We also point out key open questions to stimulate future work.

Background

Cardiac alternans are proarrhythmic and mechanistically link cardiac mechanical dysfunction and sudden cardiac death. Beat-to-beat alternans occur when beats with large Ca(2+) transients and long action potential duration (APD) alternate with the converse. APD alternans are typically driven by Ca(2+) alternans and sarcoplasmic reticulum (SR) Ca(2+) release alternans. But the effect of intercellular communication via gap junctions (GJ) on alternans in the intact heart remains unknown.

Objective

We assessed the effects of cell-to-cell coupling on local alternans in intact Langendorff-perfused mouse hearts, measuring single myocyte [Ca(2+)] alternans synchronization among neighboring cells, and effects of β-adrenergic receptor (β-AR) activation and reduced GJ coupling.

Methods and results

Mouse hearts (C57BL/6) were retrogradely perfused and loaded with Fluo8-AM to record cardiac myocyte [Ca(2+)] in situ with confocal microscopy. Single cell resolution allowed analysis of alternans within the intact organ during alternans induction. Carbenoxolone (25 μM), a GJ inhibitor, significantly increased the occurrence and amplitude of alternans in single cells within the intact heart. Alternans were concordant between neighboring cells throughout the field of view, except transiently during onset. β-AR stimulation only reduced Ca(2+) alternans in tissue that had reduced GJ coupling, matching effects seen in isolated myocytes.

Conclusions

Ca(2+) alternans among neighboring myocytes is predominantly concordant, likely because of electrical coupling between cells. Consistent with this, partial GJ uncoupling increased propensity and amplitude of Ca(2+) alternans, and made them more sensitive to reversal by β-AR activation, as in isolated myocytes. Electrical coupling between myocytes may thus limit the alternans initiation, but also allow alternans to be more stable once established.

Background

A hallmark of heart failure is impaired cytoplasmic Ca(2+) handling of cardiomyocytes. It remains unknown whether specific alterations in nuclear Ca(2+) handling via altered excitation-transcription coupling contribute to the development and progression of heart failure.

Methods and results

Using tissue and isolated cardiomyocytes from nonfailing and failing human hearts, as well as mouse and rabbit models of hypertrophy and heart failure, we provide compelling evidence for structural and functional changes of the nuclear envelope and nuclear Ca(2+) handling in cardiomyocytes as remodeling progresses. Increased nuclear size and less frequent intrusions of the nuclear envelope into the nuclear lumen indicated altered nuclear structure that could have functional consequences. In the (peri)nuclear compartment, there was also reduced expression of Ca(2+) pumps and ryanodine receptors, increased expression of inositol-1,4,5-trisphosphate receptors, and differential orientation among these Ca(2+) transporters. These changes were associated with altered nucleoplasmic Ca(2+) handling in cardiomyocytes from hypertrophied and failing hearts, reflected as increased diastolic Ca(2+) levels with diminished and prolonged nuclear Ca(2+) transients and slowed intranuclear Ca(2+) diffusion. Altered nucleoplasmic Ca(2+) levels were translated to higher activation of nuclear Ca(2+)/calmodulin-dependent protein kinase II and nuclear export of histone deacetylases. Importantly, the nuclear Ca(2+) alterations occurred early during hypertrophy and preceded the cytoplasmic Ca(2+) changes that are typical of heart failure.

Conclusions

During cardiac remodeling, early changes of cardiomyocyte nuclei cause altered nuclear Ca(2+) signaling implicated in hypertrophic gene program activation. Normalization of nuclear Ca(2+) regulation may therefore be a novel therapeutic approach to prevent adverse cardiac remodeling.