Calcium Handling Machinery and Sarcomere Assembly are Impaired Through Multipronged Mechanisms in Cancer Cytokine-Induced Cachexia

The precisely arranged sarcomeres are the fundamental units of striated muscle cells that produce force from ATP-dependent cross-bridge cycling between actin (thin filaments) and myosin (thick filaments) to bear load and drive movement. During excitation–contraction coupling (ECC) process, calcium (Ca²⁺) is released from the sarcoplasmic reticulum (SR) through the ryanodine receptor (RyR1), activating thin filament and enabling acto-myosin cross-bridge cycling, and hence myocyte contraction [1, 2]. The dihydropyridine receptor (DHPR) α1s subunit physically interacts with RyR1, inducing an opening of the channel as the result of an action potential [1]. Muscle relaxation follows the transfer of Ca²⁺ ions to the SR through the calcium ATPase pump SERCA1. Thus, a precise sarcomeric assembly, a proper Ca²⁺ transient, and correct interplay between these two systems is critical for primary muscle cell functions, i.e., contraction and force generation. Molecular insights in various aspects of these processes remain poorly determined. A detailed understanding of it is critical to apprehend not only muscle physiology but muscle wasting conditions as well.

Cancer triggers cachexia, which is a severe muscle wasting disorder associated with up to 80% of cancer patients [3]. Cancer cytokine-induced cachexia (CIC) is defined by ongoing involuntary loss of muscle and/or fat mass and is not reversible by common treatments [4], with an estimated high mortality rate ranges from 20% to 50% in cancer patients depending on cancer types [5]. The effective dose of cancer therapeutics, particularly chemotherapy, is calculated based on body surface area [6, 7]. Thus, CIC additionally aggravates the patient's responsiveness to therapies [8]. Tumour-released pro-inflammatory cytokines, such as tumour necrosis factor alpha (TNF-α), interferon gamma (INF-γ), interleukin 6 (IL-6), induce degradation of myofibril proteins, especially myosin heavy chain (MyHC), through NF-κB (nuclear factor ‘kappa-light-chain-enhancer’ of activated B-cells)-MuRF1 (muscle RING-finger protein-1) ubiquitin-proteasome pathway [9]. Furthermore, NF-κB downregulates the master transcription factor MyoD and thereby suppresses skeletal muscle differentiation of myogenic progenitor cells [10, 11]. Besides Murf1, other E3 ligases including Atrogin1 and UBR2 are also upregulated and modify thick filament proteins for degradation [12, 13]. Pro-inflammatory cytokines also contribute to insulin resistance and suppression of the insulin-like growth factor I (IGF1)-Akt pathway, further deteriorating the catabolic condition [14]. Moreover, upregulation of the metal-ion transporter ZRT- and IRT-like protein 14 (ZIP14) has been shown in cachectic skeletal muscles of mice and in human patients with metastatic cancer in response to cancer-induced TNF-α and TGF-β cytokine release [15]. Despite all these important findings, no effective treatment regimens to target CIC in human are currently available. It points toward previously undetermined molecular mechanisms that are linked with CIC.

In the present study, we observed a loss of muscle cell function, in both skeletal and cardiac muscle cells, in CIC. At physiological level, we identified a deregulated calcium homeostasis and complete disorganization of sarcomeric structures in CIC. Our system-wide approach showed that CIC reorients the transcriptional state of distinct major muscle-specific genes critical for calcium homeostasis and muscle contraction. Furthermore, our investigation unravelled chromatin-related events of distinct muscle-specific genes, as the initial trigger of CIC.

In the current study, we demonstrate the multi-pronged regulatory mechanisms underlying CIC. Through these mechanisms, primarily calcium transient pathway and sarcomere organization was found affected. One aspect of CIC was a more direct effect on protein levels of critical muscle-specific regulators, including RyR1. We reason that a significant reduction in RyR1 protein levels in CIC, served as a major cause for a strongly blunted release of Ca²⁺ from the SR. Apart from Ca²⁺ release, Ca²⁺ reuptake was also found compromised as a result of reduced levels of Ca²⁺ pump SERCA. The transcriptomic analysis revealed that the effect on SERCA1 level is aggravated by the reduced gene expression. Besides, deregulated epigenetic and transcriptional mechanisms led to downregulation of genes, particularly Myh1, Cacna1s, and Atp2a1 etc. in CIC. These multiple aspects of regulation ultimately culminated in impaired Ca²⁺ handling and loss of functional sarcomere structure leading to dysfunctional muscle cells that lacked contractile ability.

Cachexia is a whole-body metabolic syndrome that remains poorly understood. CIC affects skeletal muscle in a plethora of ways, by deregulating energy homeostasis [33], catabolism/anabolism [34], and gene expression [35, 36] among others. A detailed mechanism by which muscle Ca²⁺ homeostasis is affected in CIC was unclear. Our proteomic data showed that RyR1 is targeted in CIC with an approximately 80% reduction at protein level. This, most likely, blocked Ca²⁺ release during ECC. Our Ca²⁺ transient experiment in presence of caffeine strongly supports this notion. Whereas caffeine, an agonist of RyR1 [24], led to a steep surge in Ca²⁺ release in control cells, the same resulted in no significant response in CIC. Furthermore, we provide evidence that neither depleted Ca²⁺ stores, nor leaky RyR1 channels contribute to impaired Ca²⁺ release in CIC (Figure S2C-D and Figure S7). In addition to RyR1, DHPR expression was also down regulated in CIC (Figure 4C). The transverse tubules are highly organized sarcolemmal invaginations important for EC coupling. Here, four DHPR heterotetramers interact in a highly ordered manner with every other RyR1 homo-tetramer located in the membrane of terminal cisternae [37]. Disturbing this stoichiometry might severely affect the transduction of action potentials toward the RyR1 and thereby Ca²⁺ release. Interestingly, in our study, the DHPR subunit α1s, which physically interacts with and activates RyR1 during EC coupling [1], was also reduced on protein level in CIC. Importantly, proper function of SERCA1 ensures rapid uptake of released Ca²⁺ into the SR during a single Ca²⁺ transient event. Thus, the key function of maintaining Ca²⁺ homeostasis by SERCA1 is crucial for muscle function and health. Our study revealed that Ca²⁺ reuptake to the SR is blunted in CIC, measured by longer single exponential decay (τ) of intracellular Ca²⁺ (Figure 2E). Taken together, our study showed that deregulation of multiple components of Ca²⁺ handling machinery is one of the key mechanisms prevalent in CIC. These components include DHPR, RyR1, as well as SERCA1 pump.

RyR1 depletion is a hallmark of several myopathies [26, 38, 39]. However, these reports did not investigate the role of RyR1 and SR Ca²⁺ handling in the context of cancer secreted cytokines (TNFα and IFNγ)-induced cachexia. Here, we have demonstrated previously undetermined multipronged regulatory mechanisms underlying CIC. These mechanisms primarily target calcium transient pathway and sarcomere organization. In the context of RyR1, CIC predominantly altered RyR1 at protein level. Regarding MyHC-IId and SERCA1, CIC altered chromatin signalling on the promoters of these genes (Myh1, Atp2a1) leading to reduced active transcription state and consequently lowered protein level of these regulators. The chromatin signalling includes changes in distinct epigenetic marks (H3K4 methylation) and impaired loading of transcriptionally active RNA Pol II on these genes (Myh1, Atp2a1) under CIC condition. Through these molecular mechanisms, CIC affects functionally related pathways in skeletal muscle cells including sarcomere organization and calcium transient process, ultimately impinged on muscle cell contraction. Our result further highlighted the complex nature of aetiology of cachexia.

The implications of higher intracellular Ca²⁺ are far-reaching, as it is an important signalling molecule for many biological processes. During EC coupling, mitochondria take up Ca²⁺ ions that are released from the SR resulting in an increased ATP synthesis to fuel the energy demands of a contracting cell [40]. A disrupted Ca²⁺ cross-talk between these organelles can have severe effects and contribute to the energetic imbalance commonly observed in CIC. Interestingly, we identified several upregulated proteins involved in redox homeostasis. On the other hand, several subunits of the mitochondrial respiratory chain complex I, NADH dehydrogenase, were specifically showing reduced protein levels, indicating a possible functional impediment of mitochondrial dynamics.

A prominent finding in this study is that, apart from direct effect on protein homeostasis, CIC suppresses the gene expression at the transcriptional level for distinct muscle-specific genes, including Myh1, Myh2, Atp2a1, and Cacna1s etc. Mechanistically, CIC strongly precluded binding of transcriptionally active species of RNA Pol II on Myh1 and Atp2a1 promoters, indicating CIC can potentially affect active transcriptional processes. Further analysis revealed reduced levels of active epigenetic marker, i.e., histone modification at H3K4me3 on Myh1 and Atp2a1 promoters in CIC. Thus, it is conceivable that reduced level of H3K4me3 is the upstream event of impaired occupancy of active RNA Pol II. This notion is in line with previous findings that active RNA Pol II co-occupies chromatin domain enriched in H3K4me3 marks [30]. Specifically, the recruitment of RNA Pol II and H3K4me3 appears to be deregulated in CIC for the muscle-specific genes Myh1 and Atp2a1, as Ser2-ph and Ser5-ph RNA Pol II protein level and global H3K4me3 signal remain largely unaltered (Figure 7C,E). At present, it is unclear if any further upstream event modulates H3K4me3 marks on muscle-specific genes. One attractive hypothesis is functional alteration of histone methyltransferases, including SET1/MLL complex that catalyses H3K4me3 marks, in CIC. Additionally, we identified a putative role of SENP3 and SENP7 in Atp2a1 regulation (Figure S8). Collectively, these data explained the mechanism that lowered transcriptional output of muscle-specific genes in CIC, ultimately resulting in sarcomere disorganization and deregulated Ca²⁺ homeostasis.

In our current study, we used murine satellite cell derived primary muscle cells, primary neonatal rat cardiomyocytes and mouse C2C12 progenitor cell derived mature muscle cells (myotubes). Although these model systems served a key role in dissecting a detailed molecular mechanism underlying CIC, in vivo validation in cachectic patient biopsies or animal models needs to be addressed in futures studies. These combined strategies might help us to further comprehend the complexity of cachexia.

In conclusion, the results presented herein place the sarcomere contractile machinery and SR as important cellular compartments predominantly affected in cancer cytokine-induced muscle wasting. The coordinated action of SR and sarcomere, primarily determines the muscle's force generating and load-bearing function. A common denominator of this regulation is calcium signalling. Our study offered an understanding of intricate mechanisms by which SR-related Ca²⁺ handling is affected in CIC. This knowledge provides a framework for future studies addressing questions to ameliorate CIC by particularly modulating calcium homeostasis mechanisms in striated muscle cells.

All the experimental procedures were performed under the ethical approval of the Italian Ministry of Health and the Institutional Animal Care and Use Committee (authorization no. 83/2019-PR and N. 127/2012-A). The authors certify that they comply with the ethical guidelines for publishing in the Journal of Cachexia, Sarcopenia and Muscle: update 2019 [41].

The authors declare no conflicts of interest.