Circadian regulation of cardiac muscle function and protein degradation

Abstract

The circadian clock plays a fundamental role in physiology. In particular, the heart is a target organ where the clock orchestrates various aspects of cardiac function. At the molecular level, the clock machinery governs daily rhythms of gene expression. Such circadian regulation is in tune with the dynamic nature of heart structure and function, and provides the foundation for chronotherapeutic applications in cardiovascular diseases. In comparison, a regulatory role of the clock in cardiac protein degradation is poorly documented. Sarcomere is the structural and functional unit responsible for cardiac muscle contraction, and sarcomere components are closely regulated by protein folding and proteolysis. Emerging evidence supports a role of the circadian clock in governing sarcomere integrity and function. Particularly, recent studies uncovered a circadian regulation of a core sarcomere component TCAP. It is possible that circadian regulation of the cardiac muscle protein turnover is a key regulatory mechanism underlying cardiac remodeling in response to physiological and environmental stimuli. While the detailed regulatory mechanisms and the molecular links to cardiac (patho)physiology remain to be further studied, therapeutic strategies targeting circadian control in the heart may markedly enhance intervention outcomes against cardiovascular disease.

Introduction

Harmonization of internal timing and environmental changes is critical for fitness and wellbeing (Bass & Lazar, 2016). The circadian clock has evolved in most organisms to function at this interface, anticipating and responding to external stimuli via concerted regulation of tissue gene expression and functions (Figure 1). The cellular oscillator is the workhorse in our circadian clock system, driving downstream gene expression directly or indirectly via clock-controlled regulatory factors (Takahashi, 2017). This control by the oscillators is tissue-specific, comprehensive and integrative. First, there is very little overlap of clock-controlled genes between different tissues (~10%) (Panda, Antoch et al., 2002; Zhang, Lahens et al., 2014), underscoring a dynamic nature of circadian control with spatiotemporal precision. Second, the cellular oscillators are also synchronized at the organismal level, chiefly by the master pacemaker located at the suprachiasmatic nuclei in the hypothalamus (Mohawk, Green et al., 2012). Recent profiling studies have revealed that a vast majority of genes are controlled by the clock in at least one tissue/locale (Zhang, Lahens et al., 2014; Mure, Le et al., 2018), highlighting a collectively predominant role of the clock at the organism level. Finally, the mode of regulation of downstream genes is highly integrative, involving virtually all known steps in the gene regulation cascade, including transcription, RNA processing, translation and protein degradation (Kojima, Shingle et al., 2011; Takahashi, 2017; Partch, 2020). Overall, the picture that has emerged is a complex yet highly sophisticated biological timer that play fundamental roles in physiology.

Figure 1.

The mammalian circadian oscillator and heart function. The ubiquitous cell-autonomous circadian oscillator is the functional unit of the mammalian clock network. It consists of coupled transcription-translation negative feedback loops, including the core loop (CLOCK, NPAS2, BMAL1, PERs, CRYs) and auxiliary loops including the stabilization loop (REV-ERBs and RORs). Various regulatory factors (e.g., FBXL3/FBXL21) regulate the expression and activities of these core clock components. The oscillators drive cell-specific expression of clock-controlled genes (CCGs) in various tissues throughout the body. Heart is a key target organ of the circadian clock, and various cardiac functions are known to display oscillatory patterns and regulated by the clock.

The current review, as part of the Special Issue on “Cardiovascular Research and the Arrival of Circadian Medicine”, aims to summarize key aspects of circadian regulation of cardiac function, review recent studies on an emerging role of the clock in cardiac protein degradation, and discuss chronotherapeutic strategies to highlight the translational impact of circadian rhythms.

Circadian control for cardiovascular functions

The clock is known to play a key role in the cardiovascular system, particularly in the heart (Figure 1) (Durgan & Young, 2010; Paschos & FitzGerald, 2010; Martino & Young, 2015; Tsimakouridze, Alibhai et al., 2015; Smolensky, Hermida et al., 2020; Thosar & Shea, 2021). It is well known that various components and functions of the cardiovascular system display circadian or diurnal patterns, including heart rate, blood pressure, endothelial function, QT interval, vascular contractility, etc. Likewise, adverse cardiovascular disease (CVD) events are also time-of-the-day dependent, including morning peaks for cerebral and ischemic strokes (Smolensky, Hermida et al., 2020).

Both human and mouse studies have provided supportive evidence for a causal role of circadian disruption in cardiovascular dysfunction. Shift work, associated with chronic circadian misalignment and sleep dysregulation, has been found to increase risks in various metabolic and cardiovascular disorder (Kervezee, Kosmadopoulos et al., 2020; Schilperoort, Rensen et al., 2020). In a small human study under an acute (10-day), controlled laboratory condition where an unnatural 28-hr day schedule is imposed, enrolled subjects were found to exhibit abnormalities in a variety of physiological parameters related to metabolic, endocrine and cardiovascular functions, including a small but statistically significant increase in average arterial pressure (Scheer, Hilton et al., 2009). In accordance, mice subjected to jet-lag paradigms are prone to adverse immune and physiological consequences, including early mortality (Davidson, Sellix et al., 2006; Inokawa, Umemura et al., 2020). In a recent study (Ramsey, Stowie et al., 2020), mice undergoing a weekly phase advance in light:dark cycles followed by an occlusion/reperfusion procedure were found to have aggravated stroke outcomes, supporting a contributory role of circadian disruption in stroke risk.

Genetic studies of mouse mutants harboring clock gene disruption provide further evidence for a key role of the clock in cardio-metabolic regulation (Curtis, Cheng et al., 2007; Anea, Zhang et al., 2009; Durgan & Young, 2010). Global disruption of the core clock genes (Clock and Bmal1) led to loss of blood pressure rhythm and altered functions of the sympathetic nervous system (Curtis, Cheng et al., 2007). When Bmal1 is deleted in an adult cardiomyocyte-specific manner (Schroder, Lefta et al., 2013), these mice (iCSΔBmal1) showed dysregulated heart rate and a propensity to suffer arrhythmia. Molecular studies further revealed that the Scn5 gene, encoding the major cardiac voltage-gated sodium channel Na(V)1.5 is a clock-controlled gene with robust oscillation under normal conditions (Schroder, Lefta et al., 2013). Scn5 oscillation was dampened in iCSΔBmal1 mice, which likely plays a significant role in the observed arrhythmia risk in these mice. Recently, an anti-arrhythmic drug targeting Na(V)1.5 has recently been shown to alter circadian rhythms, although the mechanism remains to be investigated (Han, Wirianto et al., 2021).

At the molecular level, a number of studies have revealed circadian gene regulatory mechanisms underlying cardiovascular functions (Martino & Young, 2015; Xu, Jain et al., 2020). For example, analysis of the circadian mutant mice Cry−/− where both Cry1 and Cry2 were disrupted revealed a salt-sensitive hypertension phenotype, characterized by an exaggerated production of aldosterone. Molecular analysis revealed that the rate-limiting enzyme Hsd3b6 is controlled by the clock at the transcription level and remained high when Cry and circadian rhythms are ablated (Doi, Takahashi et al., 2010). In a different approach, circadian oscillation of regulatory genes can also lead to molecular insights into clock function in the cardiovascular system (Jeyaraj, Haldar et al., 2012; Rotter, Grinsfelder et al., 2014). For example, the transcription factor KLF15 was initially found to be encoded by a clock-controlled gene in the heart, and in turn regulates the rhythmic expression of KChIP2, encoding a key component of the voltage-gated potassium channel important for cardiac repolarization and ventricular arrhythmia (Jeyaraj, Haldar et al., 2012). At the global level, transcriptomic profiling studies have revealed the oscillatory gene set in the heart in both nocturnal and diurnal species, ranging between 6%-13% with distinct phase distributions (Storch, Lipan et al., 2002; Martino, Arab et al., 2004; Zhang, Lahens et al., 2014; Mure, Le et al., 2018). More in-depth functional and mechanistic studies of additional oscillatory genes in cardiac and vascular tissues will surely lead to novel insights on circadian cardiovascular regulation.

An emerging role of the clock in cardiac protein degradation

Proteostasis, including protein synthesis, processing/folding and degradation, is an important cellular mechanism in cardiac muscles (McLendon & Robbins, 2015; Henning & Brundel, 2017). Compared with non-muscle cells, cardiac muscles are terminally differentiated, must contract throughout lifetime, require robust metabolic/stress responses and involve specialized cellular machineries for electric conductance. The structural and functional unit of striated muscles, including both cardiac and skeletal muscles, is the sarcomere (Ono, 2010; Martin & Kirk, 2020), which is highly conserved throughout from worms to mammals. Sarcomeres line up sequentially, and tied together by a complex protein assembly called Z-disc to form contractible myofibrils, which in turn are bound in bundles to form cardiomyocytes. The sarcomere consists mainly of the myosin thick filaments and actin thin filaments, with a large number of associated structural and regulatory proteins. Given the heart is the first organ to be formed after birth and must continuously function until death, and that cardiomyocytes are post-mitotic, protein quality control at the sarcomere plays a particularly important role in cardiac proteostasis (Henning & Brundel, 2017; Martin & Kirk, 2020). Of particular importance is protein degradation mechanisms to remove misfolded or faulty proteins.

Protein degradation machineries in cardiomyocytes include protein degradation machineries, including calpains, the ubiquitin-proteasome system and autophagy (Portbury, Willis et al., 2011; Martin & Kirk, 2020). Calpains are a family of conserved calcium-dependent cysteine proteases, and cardiomyocytes express calpain 1 and 2, each shown to target multiple myofilament proteins (Wang, Chen et al., 2018). There is evidence that calpain-mediated proteolysis at the sarcomere lies upstream of the ubiquitin-mediated protein degradation (Galvez, Diwan et al., 2007). The ubiquitin-mediated protein degradation by proteasomes is a key protein degradation pathway in cardiomyocytes (Portbury, Willis et al., 2011). Ubiquitinated proteins, following ubiquitination reactions by the E3 ligases, are degraded by the 26S proteasome believed to be tethered to the sarcomere Z-disc (Rudolph, Huttemeister et al., 2019). Since E3 ligases are responsible for target specificity, a number of E3 ligases have been identified to mediate sarcomere protein turnover (Martin & Kirk, 2020), including the muscle RING finger E3 ligase (MuRFs), TRIM32, MDM2, FBXL22, atrogin-1 etc. For example, MuRF-1 and MuRF-2 are localized to the sarcomere Z-disc and M-line, share functional redundancy to degrade a large number of sarcomere proteins such as troponins, TCAP, beta-MHC, and myosin light chain 2 (Kedar, McDonough et al., 2004; Witt, Granzier et al., 2005; Witt, Witt et al., 2008; Chen, Czernuszewicz et al., 2012). Finally, autophagy is a cellular process to engulf and recycle cytosol fractions, organelles and protein aggregates in bulk (Nakai, Yamaguchi et al., 2007; Henning & Brundel, 2017). Cardiac tissues have been shown to undergo autophagy remodeling of the sarcomere, and it has been postulated that autophagy during sleep functions to promote repair and remodeling when the cardiac demand subsides (Rotter & Rothermel, 2012). Together, these protein degradation mechanisms are essential for sarcomere rejuvenation.

While pioneering studies have shown that several sarcomere proteins display long half-lives, ranging from days to weeks (Martin, Rabinowitz et al., 1977; Martin, 1981), evidence is emerging pointing to a circadian control of sarcomere protein turnover. For example, proteomic and in silico approaches identified diurnal oscillation and circadian control of several heart-enriched proteins with structural and regulatory roles in the sarcomere and myofilaments (Podobed, Pyle et al., 2014; Podobed, Alibhai et al., 2014; Seo, Yoon et al., 2020). The Z-disc, in addition to its structural role, serves as a signaling hub for cardiomyocyte function and disease (Frank & Frey, 2011). One interesting example is the disease relevance and integrative control of TCAP. TCAP, also known as titin-cap or telethonin, is a small, highly conserved, and abundant core component of the sarcomere Z-disc (Valle, Faulkner et al., 1997). TCAP tethers titin through its N-terminal beta sheet to the Z-disc (Gregorio, Trombitas et al., 1998; Mues, van der Ven et al., 1998), thus playing an important role in sarcomere integrity. Human TCAP mutations have been linked with the severe autosomal recessive limb-girdle muscular dystrophy (AR LGMD) type 2G and dilated cardiomyopathy; accordingly, animal models of TCAP deficiency showed various defects in the structure and function of the sarcomere (Moreira, Wiltshire et al., 2000; Knoll, Hoshijima et al., 2002; Hershberger, Parks et al., 2008; Zhang, Yang et al., 2009; Markert, Meaney et al., 2010; Knoll, Linke et al., 2011; Hirtle-Lewis, Desbiens et al., 2013; Ibrahim, Siedlecka et al., 2013; Candasamy, Haworth et al., 2014; Francis, Sunitha et al., 2014; Chamova, Bichev et al., 2018). For example, it was found that Tcap knockout mice adapt poorly to pressure overload, which ultimately leads to heart failure (Knoll, Linke et al., 2011). Both transcript and protein expressions of TCAP were found to subjected to circadian control, in part mediated by the E-box element at the transcription level (Podobed, Alibhai et al., 2014). Likewise, Tcap transcription in skeletal muscle was also found to be regulated by the circadian transcription factors CLOCK/BMAL1, in collaboration with MyoD (Hodge, Zhang et al., 2019).

Our recent study reveals a GSK-3β-FBXL21 pathway that governs circadian oscillation of TCAP protein degradation. FBXL21, along with its paralog FBXL3, were previously found to govern rhythmic degradation of the core clock components CRY1/2 in an antagonistic mechanism where FBXL21 degrades CRYs in the cytoplasm but plays a protective role by antagonizing FBXL3-mediated CRY turnover in the nucleus (Hirano, Yumimoto et al., 2013; Yoo, Mohawk et al., 2013). TCAP was identified as another cytoplasmic target substrate of FBXL21 in both cardiac and skeletal muscles (Wirianto, Yang et al., 2020). It was further shown that GSK-3β serves as an important upstream kinase that phosphorylate both FBXL21 and TCAP, regulating SCF complex formation in the proteasomal degradation pathway (Figure 2). In the hypomorphic Fbxl21 mutant mice Pasttime (Psttm), the normal circadian turnover of TCAP was dysregulated. Consistently, skeletal myofiber width was reduced, and several parameters of muscle function were impaired, including grip strength, skeletal muscle atrophy, exercise tolerance which reflects the combined skeletal and cardiac muscle function, and cardiac output function by echocardiography. Together, these observations support a pivotal role of the GSK-3β-FBXL21 axis in circadian regulation of TCAP degradation and muscle function. It should be noted that several other TCAP E3 ligases have previously been identified, including MuRFs and MDM2 (Witt, Granzier et al., 2005; Tian, Li et al., 2006; Witt, Witt et al., 2008). However, TCAP degradation does not require the ring finger domain of MDM2, and a direct mechanistic function of MuRFs in TCAP ubiquitination and degradation, despite their interaction shown by yeast 2-hybrid assays, remains to be determined.

Figure 2.

Protein degradation in the heart and circadian regulation of TCAP. Cardiac protein degradation is regulated by calpains, proteasome-mediated protein degradation and autophagy. Protein abundance of various components of the sarcomeres, containing myosin thick filaments, actin thin filaments, and Z-disc, is regulated by these pathways. In particular, the sarcomere component TCAP displays robust oscillatory patterns over the circadian cycle. Recent studies reveal that both transcript and protein expressions of TCAP are controlled by the clock, the latter involving a GSK-3β-FBXL21 axis.

The circadian regulation of TCAP is consistent with the notion that sarcomere function is a clock-controlled output. In addition to GSK-3β, several other kinases have also been implicated in sarcomere protein modification and contractile function, including PKA, PKD, CamKII and calcineurin (Bray, Shaw et al., 2008; Sachan, Dey et al., 2011; Alibhai, Tsimakouridze et al., 2014; Rotter, Grinsfelder et al., 2014). Notably, PKD and CamKII have also been shown to phosphorylate TCAP in cardiomyocytes in vitro, highlighting the mechanistic complexity of circadian regulation of sarcomere components.

Circadian time-based therapeutic strategies for CVD

Disease conditions and adverse events often manifest in a circadian time-dependent manner; likewise, medications are also known to act (dynamics) or be metabolized (kinetics) inside our body according to circadian rhythmicity (Levi & Schibler, 2007; Smolensky, Hermida et al., 2020). Therefore, chronotherapy, namely temporally controlled administration of medications or therapies, has been studied for CVD, cancer, metabolic disease and other clock-related disorders (Levi & Schibler, 2007; Zhang, Lahens et al., 2014; Sundar, Yao et al., 2015; Tsimakouridze, Alibhai et al., 2015; Smolensky, Hermida et al., 2020). While daytime blood pressure measurements have traditionally been used as the clinical standard for evaluating hypertension risk, strong evidence indicates that the ambulatory blood pressure monitoring (ABPM) is most informative and accurate for CVD risk assessment (Hermida, Crespo et al., 2018; Smolensky, Hermida et al., 2020). Particularly, based on the dipping pattern in blood pressure at nighttime, the non-dippers and risers, with much reduced dipping or even with greater blood pressure when asleep, are at the highest CVD risk, and medications should aim to normalize sleep-time systolic blood pressure (SBP). Despite the prevalence of circadian gene expression and the fact that most top drugs target proteins encoded by clock-controlled genes (Zhang, Lahens et al., 2014; Mure, Le et al., 2018), less than 1% of marketed drugs in the US have clear designation of preferred circadian time (Smolensky, Hermida et al., 2020). This is clearly an urgent unmet need. Further supporting the need to take into account the circadian timing in disease treatment, a randomized clinical study recently found that patients undergoing aortic valve replacement surgery show distinct clinical outcome depending on whether the surgery was performed in the morning or afternoon, with those receiving afternoon surgery displaying significantly lower risk of major adverse events (Montaigne, Marechal et al., 2018). Although the mechanism remains to be further investigated, this study illustrates the utility of chronotherapy for surgical interventions.

As opposed to altering timing of existing therapy, manipulation of circadian timing is a complementary strategy to elicit beneficial effects. Such manipulations can include behavioral, dietary and chemical means (Schroeder & Colwell, 2013; Wallach & Kramer, 2015; Chen, Yoo et al., 2018). Other than exercise (Henning & Brundel, 2017), another promising life-style intervention that has emerged in recent years is time-restricted feeding/eating (TRF/TRE), namely limiting food availability/eating time to a time window (typically 6-10 hrs) early during the active period (Hatori, Vollmers et al., 2012; Melkani & Panda, 2017). It has been shown that in fruit flies, TRF was able to protect age- and high-fat diet-induced cardiac and mitochondrial decline (Gill, Le et al., 2015) and improve skeletal muscle function and sarcomere organization (Villanueva, Livelo et al., 2019). Effects of TRF/TRE in humans are being actively investigated, and there is evidence indicating improvement of cardiometabolic parameters in randomized trials (Cienfuegos, Gabel et al., 2020; Gabel, Cienfuegos et al., 2021). Clock-modulating compounds have shown promising effects to counteract age-related decline and metabolic/immune dysfunctions relevant to cardiovascular health (Billon, Sitaula et al., 2016; Zhang, Zhang et al., 2017; Nohara, Nemkov et al., 2019; Reitz, Alibhai et al., 2019). Interestingly, just one-time dosing of the REV-ERB agonist SR9009 was able to diminish heart failure risk in mice post-myocardial ischemia reperfusion (Reitz, Alibhai et al., 2019). Finally, a number of drugs have been investigated to improve cardiac proteostasis in both animal models and in humans (Henning & Brundel, 2017), although their possible circadian effects remain to be investigated.

Concluding remarks

CVD remains a leading medical challenge globally. As detailed above, circadian clocks are a particularly important biological mechanism and interventional target for CVD. Despite encouraging progresses in understanding circadian regulatory mechanisms and physiological bases underlying certain cardiovascular (dys)functions, clearly much remains to be investigated in preclinical and clinal settings. As the structural unit and signaling hub in cardiomyocytes, the sarcomere is highly dynamic under various conditions and in response to stresses, and the circadian clock plays an integral role in maintaining sarcomere structure and function. While most CVD events and drug targets are clearly clock-related, most standard of care practices have not incorporated chronotherapeutic design, often for pragmatic reasons. Clearly progress in this area should be made to enhance intervention outcomes. In addition, time manipulation by behavioral, dietary, and pharmacological approaches is a relative nascent approach which should be further explored for CVD and other clock-related diseases.