Nuclear histone deacetylase 4 (HDAC4) represses MEF2-mediated transcription, implicated in the development of heart failure. CaMKII-dependent phosphorylation drives nucleus-to-cytoplasm HDAC4 shuttling, but protein kinase A (PKA) is also linked to HDAC4 translocation. However, the interplay of CaMKII and PKA in regulating adult cardiomyocyte HDAC4 translocation is unclear. Here we sought to determine the interplay of PKA- and CaMKII-dependent HDAC4 phosphorylation and translocation in adult mouse, rabbit and human ventricular myocytes. Confocal imaging and protein analyses revealed that inhibition of CaMKII-but not PKA, PKC or PKD-raised nucleo-to-cytoplasmic HDAC4 fluorescence ratio (F_Nuc/F_Cyto) by ~ 50%, indicating baseline CaMKII activity that limits HDAC4 nuclear localization. Further CaMKII activation (via increased extracellular [Ca²⁺], high pacing frequencies, angiotensin II or overexpression of CaM or CaMKIIδC) led to significant HDAC4 nuclear export. In contrast, PKA activation by isoproterenol or forskolin drove HDAC4 into the nucleus (raising F_Nuc/F_Cyto by > 60%). These PKA-mediated effects were abolished in cells pretreated with PKA inhibitors and in cells expressing mutant HDAC4 in S265/266A mutant. In physiological conditions where both kinases are active, PKA-dependent nuclear accumulation of HDAC4 was predominant in the very early response, while CaMKII-dependent HDAC4 export prevailed upon prolonged stimuli. This orchestrated co-regulation was shifted in failing cardiomyocytes, where CaMKII-dependent effects predominated over PKA-dependent response. Importantly, human cardiomyocytes showed similar CaMKII- and PKA-dependent HDAC4 shifts. Collectively, CaMKII limits nuclear localization of HDAC4, while PKA favors HDAC4 nuclear retention and S265/266 is essential for PKA-mediated regulation. These pathways thus compete in HDAC4 nuclear localization and transcriptional regulation in cardiac signaling.

Rationale

The mechanisms underlying atrial fibrillation (AF), the most common clinical arrhythmia, are poorly understood. Nucleoplasmic Ca²⁺ regulates gene expression, but the nature and significance of nuclear Ca²⁺-changes in AF are largely unknown.

Objective

To elucidate mechanisms by which AF alters atrial-cardiomyocyte nuclear Ca²⁺ ([Ca²⁺]_Nuc) and CaMKII (Ca²⁺/calmodulin-dependent protein kinase-II)-related signaling.

Methods and results

Atrial cardiomyocytes were isolated from control and AF dogs (kept in AF by atrial tachypacing [600 bpm × 1 week]). [Ca²⁺]_Nuc and cytosolic [Ca²⁺] ([Ca²⁺]_Cyto) were recorded via confocal microscopy. Diastolic [Ca²⁺]_Nuc was greater than [Ca²⁺]_Cyto under control conditions, while resting [Ca²⁺]_Nuc was similar to [Ca²⁺]_Cyto; both diastolic and resting [Ca²⁺]_Nuc increased with AF. IP₃R (Inositol-trisphosphate receptor) stimulation produced larger [Ca²⁺]_Nuc increases in AF versus control cardiomyocytes, and IP₃R-blockade suppressed the AF-related [Ca²⁺]_Nuc differences. AF upregulated nuclear protein expression of IP₃R1 (IP₃R-type 1) and of phosphorylated CaMKII (immunohistochemistry and immunoblot) while decreasing the nuclear/cytosolic expression ratio for HDAC4 (histone deacetylase type-4). Isolated atrial cardiomyocytes tachypaced at 3 Hz for 24 hours mimicked AF-type [Ca²⁺]_Nuc changes and L-type calcium current decreases versus 1-Hz-paced cardiomyocytes; these changes were prevented by IP₃R knockdown with short-interfering RNA directed against IP₃R1. Nuclear/cytosolic HDAC4 expression ratio was decreased by 3-Hz pacing, while nuclear CaMKII phosphorylation was increased. Either CaMKII-inhibition (by autocamtide-2-related peptide) or IP₃R-knockdown prevented the CaMKII-hyperphosphorylation and nuclear-to-cytosolic HDAC4 shift caused by 3-Hz pacing. In human atrial cardiomyocytes from AF patients, nuclear IP₃R1-expression was significantly increased, with decreased nuclear/nonnuclear HDAC4 ratio. MicroRNA-26a was predicted to target ITPR1 (confirmed by luciferase assay) and was downregulated in AF atrial cardiomyocytes; microRNA-26a silencing reproduced AF-induced IP₃R1 upregulation and nuclear diastolic Ca²⁺-loading.

Conclusions

AF increases atrial-cardiomyocyte nucleoplasmic [Ca²⁺] by IP₃R1-upregulation involving miR-26a, leading to enhanced IP₃R1-CaMKII-HDAC4 signaling and L-type calcium current downregulation. Graphic Abstract: A graphic abstract is available for this article.

Mitochondrial dysfunction in cardiomyocytes is a hallmark of heart failure development. Although initial studies recognized the importance of different mitochondrial subpopulations, there is a striking lack of direct comparison of intrafibrillar (IF) versus perinuclear (PN) mitochondria during the development of HF. Here, we use multiple approaches to examine the morphology and functional properties of IF versus PN mitochondria in pressure overload-induced cardiac remodelling in mice, and in non-failing and failing human cardiomyocytes. We demonstrate that PN mitochondria from failing cardiomyocytes are more susceptible to depolarization of mitochondrial membrane potential, reactive oxygen species generation and impairment in Ca²⁺ uptake compared with IF mitochondria at baseline and under physiological stress protocol. We also demonstrate, for the first time to our knowledge, that under normal conditions PN mitochondrial Ca²⁺ uptake shapes nucleoplasmic Ca²⁺ transients (CaTs) and limits nucleoplasmic Ca²⁺ loading. The loss of PN mitochondrial Ca²⁺ buffering capacity translates into increased nucleoplasmic CaTs and may explain disproportionate rise in nucleoplasmic [Ca²⁺] in failing cardiomyocytes at increased stimulation frequencies. Therefore, a previously unidentified benefit of restoring the mitochondrial Ca²⁺ uptake may be normalization of nuclear Ca²⁺ signalling and alleviation of altered excitation-transcription, which could be an important therapeutic approach to prevent adverse cardiac remodelling. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.

Rationale

CaMKII (Ca²⁺-Calmodulin dependent protein kinase) δC activation is implicated in pathological progression of heart failure (HF) and CaMKIIδC transgenic mice rapidly develop HF and arrhythmias. However, little is known about early spatio-temporal Ca²⁺ handling and CaMKII activation in hypertrophy and HF.

Objective

To measure time- and location-dependent activation of CaMKIIδC signaling in adult ventricular cardiomyocytes, during transaortic constriction (TAC) and in CaMKIIδC transgenic mice.

Methods and results

We used human tissue from nonfailing and HF hearts, 4 mouse lines: wild-type, KO (CaMKIIδ-knockout), CaMKIIδC transgenic in wild-type (TG), or KO background, and wild-type mice exposed to TAC. Confocal imaging and biochemistry revealed disproportional CaMKIIδC activation and accumulation in nuclear and perinuclear versus cytosolic regions at 5 days post-TAC. This CaMKIIδ activation caused a compensatory increase in sarcoplasmic reticulum Ca²⁺ content, Ca²⁺ transient amplitude, and [Ca²⁺] decline rates, with reduced phospholamban expression, all of which were most prominent near and in the nucleus. These early adaptive effects in TAC were entirely mimicked in young CaMKIIδ TG mice (6-8 weeks) where no overt cardiac dysfunction was present. The (peri)nuclear CaMKII accumulation also correlated with enhanced HDAC4 (histone deacetylase) nuclear export, creating a microdomain for transcriptional regulation. At longer times both TAC and TG mice progressed to overt HF (at 45 days and 11-13 weeks, respectively), during which time the compensatory Ca²⁺ transient effects reversed, but further increases in nuclear and time-averaged [Ca²⁺] and CaMKII activation occurred. CaMKIIδ TG mice lacking δB exhibited more severe HF, eccentric myocyte growth, and nuclear changes. Patient HF samples also showed greatly increased CaMKIIδ expression, especially for CaMKIIδC in nuclear fractions.

Conclusions

We conclude that in early TAC perinuclear CaMKIIδC activation promotes adaptive increases in myocyte Ca²⁺ transients and nuclear transcriptional responses but that chronic progression of this nuclear Ca²⁺-CaMKIIδC axis contributes to eccentric hypertrophy and HF.