Nuclear Calcium in Cardiac (Patho)Physiology: Small Compartment, Big Impact.

The nucleus of a cardiomyocyte has been increasingly recognized as a morphologically distinct and partially independent calcium (Ca2+) signaling microdomain, with its own Ca2+-regulatory mechanisms and important effects on cardiac gene expression. In this review, we (1) provide a comprehensive overview of the current state of research on the dynamics and regulation of nuclear Ca2+ signaling in cardiomyocytes, (2) address the role of nuclear Ca2+ in the development and progression of cardiac pathologies, such as heart failure and atrial fibrillation, and (3) discuss novel aspects of experimental methods to investigate nuclear Ca2+ handling and its downstream effects in the heart. Finally, we highlight current challenges and limitations and recommend future directions for addressing key open questions.

Functional remodelling of perinuclear mitochondria alters nucleoplasmic Ca2+ signalling in heart failure.

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'.

ß-Adrenergic Receptor Stimulation Maintains NCX-CaMKII Axis and Prevents Overactivation of IL6R-Signaling in Cardiomyocytes upon Increased Workload.

Excessive ß-adrenergic stimulation and tachycardia are potent triggers of cardiac remodeling; however, their exact cellular effects remain elusive. Here, we sought to determine the potency of ß-adrenergic stimulation and tachycardia to modulate gene expression profiles of cardiomyocytes. Using neonatal rat ventricular cardiomyocytes, we showed that tachycardia caused a significant upregulation of sodium-calcium exchanger (NCX) and the activation of calcium/calmodulin-dependent kinase II (CaMKII) in the nuclear region. Acute isoprenaline treatment ameliorated NCX-upregulation and potentiated CaMKII activity, specifically on the sarcoplasmic reticulum and the nuclear envelope, while preincubation with the ß-blocker propranolol abolished both isoprenaline-mediated effects. On a transcriptional level, screening for hypertrophy-related genes revealed tachycardia-induced upregulation of interleukin-6 receptor (IL6R). While isoprenaline prevented this effect, pharmacological intervention with propranolol or NCX inhibitor ORM-10962 demonstrated that simultaneous CaMKII activation on the subcellular Ca2+ stores and prevention of NCX upregulation are needed for keeping IL6R activation low. Finally, using hypertensive Dahl salt-sensitive rats, we showed that blunted ß-adrenergic signaling is associated with NCX upregulation and enhanced IL6R signaling. We therefore propose a previously unrecognized protective role of ß-adrenergic signaling, which is compromised in cardiac pathologies, in preventing IL6R overactivation under increased workload. A better understanding of these processes may contribute to refinement of therapeutic options for patients receiving ß-blockers.

miR-1183 Is a Key Marker of Remodeling upon Stretch and Tachycardia in Human Myocardium.

Many cardiac insults causing atrial remodeling are linked to either stretch or tachycardia, but a comparative characterization of their effects on early remodeling events in human myocardium is lacking. Here, we applied isometric stretch or sustained tachycardia at 2.5 Hz in human atrial trabeculae for 6 h followed by microarray gene expression profiling. Among largely independent expression patterns, we found a small common fraction with the microRNA miR-1183 as the highest up-regulated transcript (up to 4-fold). Both, acute stretch and tachycardia induced down-regulation of the predicted miR-1183 target genes ADAM20 and PLA2G7. Furthermore, miR-1183 was also significantly up-regulated in chronically remodeled atrial samples from patients with persistent atrial fibrillation (3-fold up-regulation versus sinus rhythm samples), and in ventricular myocardium from dilative cardiomyopathy hearts (2-fold up-regulation) as compared to non-failing controls. In sum, although stretch and tachycardia show distinct transcriptomic signatures in human atrial myocardium, both cardiac insults consistently regulate the expression of miR-1183 and its downstream targets in acute and chronic remodeling. Thus, elevated expression of miR-1183 might serve as a tissue biomarker for atrial remodeling and might be of potential functional significance in cardiac disease.

HDAC Inhibition Regulates Cardiac Function by Increasing Myofilament Calcium Sensitivity and Decreasing Diastolic Tension.

We recently established a large animal model that recapitulates key clinical features of heart failure with preserved ejection fraction (HFpEF) and tested the effects of the pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA). SAHA reversed and prevented the development of cardiopulmonary impairment. This study evaluated the effects of SAHA at the level of cardiomyocyte and contractile protein function to understand how it modulates cardiac function. Both isolated adult feline ventricular cardiomyocytes (AFVM) and left ventricle (LV) trabeculae isolated from non-failing donors were treated with SAHA or vehicle before recording functional data. Skinned myocytes were isolated from AFVM and human trabeculae to assess myofilament function. SAHA-treated AFVM had increased contractility and improved relaxation kinetics but no difference in peak calcium transients, with increased calcium sensitivity and decreased passive stiffness of myofilaments. Mass spectrometry analysis revealed increased acetylation of the myosin regulatory light chain with SAHA treatment. SAHA-treated human trabeculae had decreased diastolic tension and increased developed force. Myofilaments isolated from human trabeculae had increased calcium sensitivity and decreased passive stiffness. These findings suggest that SAHA has an important role in the direct control of cardiac function at the level of the cardiomyocyte and myofilament by increasing myofilament calcium sensitivity and reducing diastolic tension.

Loss of autophagy protein ATG5 impairs cardiac capacity in mice and humans through diminishing mitochondrial abundance and disrupting Ca2+ cycling.

AIMS: Autophagy protects against the development of cardiac hypertrophy and failure. While aberrant Ca2+ handling promotes myocardial remodelling and contributes to contractile dysfunction, the role of autophagy in maintaining Ca2+ homeostasis remains elusive. Here, we examined whether Atg5 deficiency-mediated autophagy promotes early changes in subcellular Ca2+ handling in ventricular cardiomyocytes, and whether those alterations associate with compromised cardiac reserve capacity, which commonly precedes the onset of heart failure. METHODS AND RESULTS: RT-qPCR and immunoblotting demonstrated reduced Atg5 gene and protein expression and decreased abundancy of autophagy markers in hypertrophied and failing human hearts. The function of ATG5 was examined using cardiomyocyte-specific Atg5-knockout mice (Atg5-/-). Before manifesting cardiac dysfunction, Atg5-/- mice showed compromised cardiac reserve in response to ß-adrenergic stimulation. Consequently, effort intolerance and maximal oxygen consumption were reduced during treadmill-based exercise tolerance testing. Mechanistically, cellular imaging revealed that Atg5 deprivation did not alter spatial and functional organization of intracellular Ca2+ stores or affect Ca2+ cycling in response to slow pacing or upon acute isoprenaline administration. However, high-frequency stimulation exposed stunted amplitude of Ca2+ transients, augmented nucleoplasmic Ca2+ load, and increased CaMKII activity, especially in the nuclear region of hypertrophied Atg5-/- cardiomyocytes. These changes in Ca2+ cycling were recapitulated in hypertrophied human cardiomyocytes. Finally, ultrastructural analysis revealed accumulation of mitochondria with reduced volume and size distribution, meanwhile functional measurements showed impaired redox balance in Atg5-/- cardiomyocytes, implying energetic unsustainability due to overcompensation of single mitochondria, particularly under increased workload. CONCLUSION: Loss of cardiac Atg5-dependent autophagy reduces mitochondrial abundance and causes subtle alterations in subcellular Ca2+ cycling upon increased workload in mice. Autophagy-related impairment of Ca2+ handling is progressively worsened by ß-adrenergic signalling in ventricular cardiomyocytes, thereby leading to energetic exhaustion and compromised cardiac reserve.

CaMKIIδC Drives Early Adaptive Ca2+ Change and Late Eccentric Cardiac Hypertrophy.

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.

The role of stretch, tachycardia and sodium-calcium exchanger in induction of early cardiac remodelling.

Stretch and tachycardia are common triggers for cardiac remodelling in various conditions, but a comparative characterization of their role in the excitation-transcription coupling (ETC) and early regulation of gene expression and structural changes is lacking. Here, we show that stretch and tachycardia directly induced hypertrophy of neonatal rat cardiac myocytes and also of non-myocytes. Both triggers induced similar patterns of hypertrophy but had largely distinct gene expression profiles. ACTA1 served as good hypertrophy marker upon stretch, while RCAN1 was found increased in response to tachycardia in a rate-dependent fashion. Mechanistically, several calcium-handling proteins, including the sodium-calcium exchanger (NCX), contributed to ETC. Phosphorylation of the calcium/calmodulin-dependent protein kinase II (CaMKII) was elevated and occurred downstream of NCX activation upon tachycardia, but not stretch. Microarray profiling revealed that stretch and tachycardia regulated around 33% and 20% genes in a NCX-dependent manner, respectively. In conclusion, our data show that hypertrophy induction by stretch and tachycardia is associated with different gene expression profiles with a significant contribution of the NCX.