Transient T-type or Low-Voltage Activated (LVA) calcium (Ca²⁺) channels are voltage-dependent ion channels that open at relatively low membrane potentials (i.e., between −70 to −60 mV, with maximum peak current between −30 to −10 mV), allowing extracellular calcium to enter the cell, and which rapidly inactivate (i.e., tau of 15–30 ms) and slowly deactivate ^[1][2][1,2]. The low threshold of activation, not far from the resting potential of most excitable cells, together with their fast kinetics, makes T-type Ca²⁺ channels key modulators of cellular excitability and pacemaking ^[2][3][2,3]. Moreover, T-type Ca²⁺ channels have a voltage range of activation that overlaps that of their steady-state inactivation, meaning that, over a small near-resting voltage range, a fraction of channels can open without completely inactivating, generating a “window” current, distributed around −60/−50 mV, that modulates intracellular calcium levels [4]. T-type Ca²⁺ channels are involved in multiple physiological processes, such as neuronal firing, nociception, electrical automaticity, blood vessel constriction and dilation, lymphatic vessel pacemaking and contraction, smooth muscle contraction, myoblasts fusion, neurotransmitter release, fertilization, cell growth, differentiation, and proliferation ^[2][5][6][7][2,5,6,7]. Thus, they are expressed in a variety of excitable and non-excitable tissues in which they display distinctive behaviors at the pharmacological and kinetic (especially in terms of inactivation) levels. This is partly due to the differential and heterogeneous expression of the following three independent T-type channel genes that encode, respectively, for the three alpha subunit subtypes named Cav3.1, Cav3.2, and Cav3.3: CACNA1G, CACNA1H, and CACNA1I ^[8][9][10][8,9,10]. Similar to other Ca²⁺ channels, like the long-lasting L-type High-Voltage Activated (HVA) ones (i.e., Cav1.x and Cav2.x), the Cav3.x alpha pore-forming subunit is organized into four domains (DI-DIV), each formed by six transmembrane segments (S1–S6), where the S4 segment contains multiple positively charged arginine or lysine residues that serve as a voltage sensor, and with the pore comprised of between segments S5 and S6. This region, responsible for selective permeability, contains four key acidic glutamate or aspartate residues [11]. In particular, the selectivity filter is determined by two glutamate residues in domains I and II and two aspartate residues in domains III and IV, a structure that differs from the one composed of four glutamate residues found in HVA Ca²⁺ channels [12]. In contrast to Cav1.x and Cav2.x, Cav3.x alpha channels do not have either the alpha-interaction domain (AID), an 18-residue sequence in the I-II intracellular linker loop necessary for the interaction with the beta subunit and conserved among all HVA Ca²⁺ channels, nor the IQ calmodulin-binding motif (“IQ” derives from the first two conserved residues of the motif itself) located in the cytoplasmic C-terminal tail and which binds calmodulin. Moreover, LVA Ca²⁺ channels do not seem to co-assemble with ancillary subunits, and expression of just the Cav3.x alpha is enough to recapitulate the native T-type current waveforms [9]. Within the family of T-type Ca²⁺ channels, Cav3.1 and Cav3.2 can be distinguished from each other through their different sensitivities to nickel inhibition and by their kinetics of recovery from inactivation, while Cav3.3 is characterized by much slower kinetics of activation and inactivation ^[13][14][13,14]. Another level of complexity is given by multiple alternative splicing variants that differ at both pharmacological and electrophysiological levels [15]. Despite functional and pharmacological differences, subtype-specific experimental tools (e.g., inhibitors) are still lacking, making the study of T-type Ca²⁺ channels in native tissues and cells particularly intricate ^[3][15][3,15], but still necessary for a complete understanding of their physiological and pathophysiological role.

In the heart, T-type Ca²⁺ channels had been traditionally considered a minor player in cardiac calcium handling. In fact, the vast majority of calcium influx responsible for cardiomyocytes contraction is managed by the more abundantly expressed HVA Ca²⁺ channels. This view has developed over the last 30 years, and, nowadays, cardiac T-type Ca²⁺ channels are considered key regulators of cardiac automaticity, development, and excitation-contraction coupling in several animal models, including mouse, rat, cat, pig, and dog ^[2][16][2,31]. At the cardiac level, T-type Ca²⁺ current (ICa_T) is carried mainly by the Cav3.1 and Cav3.2 sub-types ^[16][17][31,32]. Their expression in cardiac tissue reaches a maximum in embryonic development and dramatically falls in the post-neonatal phase ^[16][17][31,32]. In particular, the amount of T-type Ca²⁺ channels decrease by about 80% from the embryonic stage to adulthood ^[18][33]. During fetal development, Cav3.2 is the most abundant sub-type expressed throughout the heart ^[19][34]. In the perinatal stage, the expression of Cav3.2 starts to decrease, while Cav3.1 levels rise and become the predominant adult cardiac sub-type ^[20][35]. In the adult heart, T-type Ca²⁺ channels are not expressed in ventricular myocytes, and tend to localize in the conduction system, and in all cell types, characterized by automaticity, where they exert a pacemaker role and function in the depolarization of the sinoatrial nodal cells. The Cav3.1/Cav3.2 ratio varies between different animal models, probably underlying the distinctive heart rates of different mammalian species ^[16][31]. Moreover, an inverse correlation has been described between sinoatrial ICa_T amplitude and body size, with smaller animals exhibiting a more prominent T-type current [1]. Despite this body of evidence, ICa_T has never been directly recorded in human nodal cells ^[1][21][1,36]. On the other hand, transcripts of both Cav3.1 and Cav3.2 have been found in the human sinoatrial node ^[8][22][8,37], with only Cav3.1 detected at the protein level ^[23][38]. Some evidence suggests a functional role of ICa_T in humans, as oral administration of mibrefradil, a relatively selective T-type Ca²⁺ channel inhibitor, reduced the pacemaker activity of the sinus node ^[24][39]. Finally, T-type Ca²⁺ channels are involved in the diseased heart. Indeed, despite not being expressed in healthy adult cardiomyocytes, as already mentioned, an increase in ICa_T has been reported in several animal models of heart failure and cardiac hypertrophy ^[17][25][26][32,40,41]. A greater expression of ICa_T can lead to alteration of intracellular calcium handling, intracellular calcium accumulation, and unbalanced calcium signaling. In fact, as demonstrated by knock-out mice, Cav3.2 is involved in the cardiac hypertrophic response, either mediated by mechanical stress, pressure overload, or angiotensin II infusion ^[27][42].

T-type Ca²⁺ channels were first described in peripheral sensory neurons whose cell bodies are located in the dorsal root ganglia (DRGs) ^[28][43]. DRGs are key sites for the mechanism underlying chronic and/or neuropathic pain perception ^[29][44]. Within this context, T-type Ca²⁺ channels, and in particular the Cav3.2 sub-type, are key players in the acute nociceptive processing induced by reducing agents ^[30][45] and are also associated with chronic pain symptoms in rats with peripheral axonal injury ^[31][46]. Interestingly, Cav3.2 seems to be particularly highly expressed in a subpopulation of nociceptive, capsaicin-sensitive DRG neurons (called “T-rich”) which exhibit T-type, but not L-type, calcium currents ^[32][47]. The role of Cav3.2 in pain perception is confirmed by several studies on different pain models in which channel expression and/or activity are increased after pain-inducing treatments, such as DRG chronic compression, spinal nerve ligation, paclitaxel-induced peripheral neuropathy, and others (for review see ^[33][48]). Despite the mounting evidence of an association, the mechanism underlying the increase of Cav3.2 in pain models remains elusive. There is a general inconsistency among studies focused on changes in total protein expression, as some reported an increase while others have observed no change. More agreement is found regarding surface protein expression which is suggested to be augmented in both early and late phases of chronic pain ^[34][35][49,50]. At least, in the latter, this is thought to be related to reduced internalization, due to lower levels of ubiquitination as a direct consequence of the overexpression of the ubiquitin-specific cysteine protease 5/isopeptidase (USP5) ^[36][37][51,52], which interacts with the III-IV linker of the Cav3.2 T-type channel, enhancing its stability.

T-type Ca²⁺ channels are also involved in a specific form of peripheral neuropathy induced by chemotherapy (CIPN). CIPN is a major dose-limiting side effect of several anticancer agents, such as immunomodulatory, platinum-based drugs, vinca alkaloids, epothilones, taxanes, and proteasome inhibitors ^[38][39][53,54]. Immunomodulatory drugs (e.g., thalidomide) are used in the treatment of multiple myeloma. They induce CIPN by downregulating TNF-α and accelerate neuronal cell death. Platinum-based (e.g., oxaliplatin, cisplatin and carboplatin) antineoplastic drugs are widely used in the treatment of several types of solid tumors. Their involvement in CIPN is due to their effect, among others, on the activity of potassium channels, transient receptor potential (TRP) and voltage-gated sodium channels (Nav 1.6, 1.7 and 1.9). Indeed, an increase in Na⁺ conductance and a reduction in the threshold potential and membrane resistance result in hyperexcitability of peripheral neurons ^[39][54]. Vinca alkaloids, used in breast cancer, germ cell tumors, Hodgkin and non-Hodgkin lymphomas, osteosarcoma, and neuroblastoma, inhibit the assembly of microtubules and promote their disassembly, thus disrupting axonal transport and leading to metaphase arrest. They are known to alter the expression of ion channels ^[39][54]. Epothilones (e.g., ixabepilone), used in the treatment of breast, ovarian, prostate, and non-small cell lung cancer act as tubulin destabilizers, causing impairment of cancer cell division leading to cell death. Meanwhile, they are responsible for the impairment of axonal transport of synaptic vesicles loaded with essential cellular components, including ion channels. Taxanes (e.g., paclitaxel, docetaxel, and cabazitaxel), are used for the treatment of ovarian, breast, non-small cell lung cancer and prostate cancer ^[40][55]. Similar to epothilones, they bind to the β-tubulin subunit, stabilizing the microtubule structure and preventing depolymerization. This condition leads to the arrest of the cell cycle at the G2/M phase. Moreover, microtubule stabilization modifies the expression and function of Na⁺, K⁺, and TRP ion channels. In particular, they decrease the expression of potassium channels and increase that of sodium Nav1.7 channels, which results in the hyperexcitability of peripheral neurons.

Additionally, taxanes exert a direct effect on Cav3.2 and ICa_T. In fact, Taxol (paclitaxel), the most commonly used taxane, has a >50% probability of inducing peripheral neuropathy, which can become chronic and irreversible in a subgroup of patients ^[41][56]. Moreover, Li and colleagues showed that, in neurons isolated from rat DRGs, Taxol increased both Cav3.2 expression and ICa_T density ^[42][57]. The treatment also left-shifted both the ICa_T voltage-dependent activation and the steady-state inactivation curves, increasing the number of available channels and potentially lowering the neuronal firing threshold ^[42][57].

Another chemotherapeutic agent that causes CIPN through a direct effect on Cav3.2 is the boronic acid dipeptidase 20S proteasome complex inhibitor bortezomib (BTZ). This class of antineoplastic drug has been developed to tackle cancer, since an over-activation of the proteostatic system machinery (e.g., the ubiquitin proteasome- and the autophagy lysosome-degradation systems) is a well-known characteristic of advanced tumors ^[43][44][45][58,59,60]. By inhibiting proteasome degradation, BTZ elevates Cav3.2 protein levels and the related current in afferent neurons, leading to BTZ-induced peripheral neuropathy (BIPN). BTZ is commonly used in the treatment of multiple myeloma and mantle cell non-Hodgkin’s lymphoma ^[46][61] and exerts its therapeutic action by inducing an arrest of the cell cycle, upregulating pro-apoptotic genes, and downregulating key factors of angiogenesis, stroma adhesion, cell proliferation and survival ^[47][48][62,63].

Despite BTZ efficacy, BIPN is one of the most severe non-hematological side effects of chemotherapeutic agents against multiple myeloma ^[46][61]. The ability of BTZ to inhibit proteasome activity in DRG neurons has been demonstrated in rat and mice models of BIPN ^[49][50][64,65]. In a recent study, Tomita and colleagues showed that in a mouse model of BIPN, the protein expression of Cav3.2 and USP5 was upregulated without increasing mRNA levels, suggesting that BTZ increases Cav3.2 protein level by reducing its proteasomal degradation. In fact, BIPN was reversed by knockdown of Cav3.2 and by the administration of T-type channel blockers, including the state-dependent blocker TTA-2, the state-independent blocker PNG, the PNG-analogue KTt-45, and ascorbic acid, which selectively blocks Cav3.2 but not Cav3.1 and Cav3.3 ^[51][66]. Interestingly, another new generation proteasome inhibitor, carfilzomib (CFZ), showed minimal neurotoxicity and fewer and milder off-target effects compared to BTZ ^[52][67]. This reduced toxicity is thought to be due to higher selectivity of CFZ for the chymotrypsin-like activity of the ꞵ5 sub-unit of the 20S core particle of the proteasome ^[53][68]. On the other hand, CFZ treatment was associated with a 5% incidence of unpredictable cardiovascular events, including congestive heart failure, pulmonary edema, decreased ejection fraction, cardiac arrest, and myocardial ischemia ^[54][69]. BTZ therapy itself, though, is not without cardiac side effects responsible for therapy discontinuation ^[55][56][70,71].

The Ubiquitin-Proteasome System (UPS) is one of the major protein degradation systems in eukaryotic cells and it accounts for up to 90% of the degradation of long- and short-lived and abnormal intracellular proteins ^[57][72]. Despite the cytosolic localization of its components, the UPS can target proteins from the plasma membrane, nucleus, and even from the ER lumen ^[58][73]. The pathway through which a protein undergoes UPS degradation is composed of two distinct events: first, a chain of multiple ubiquitin molecules is covalently attached to the target protein, and second, the tagged protein is transported to the proteasome for degradation. The structure and function of the proteasome have been extensively studied and reviewed ^{[59][60][61][62][63][64]}[74,75,76,77,78,79] and go beyond the scope of this rentryview. The proteasome and the ubiquitin-activating enzymes are constitutively active. Nevertheless, UPS is finely regulated, as the ubiquitination state of a protein is a dynamic counterbalance between ubiquitination and de-ubiquitination ^[58][73]. This machinery is of course involved in the regulation of the surface expression not only of T-type but also of several ion channels. The incubation with the proteasome inhibitor MG132, a structural and functional analog of BTZ that enhances Cav3.2 activity in rat DRGs ^[36][51], extended the half-life of cardiac Kv1.5 expressed in COS cells, inducing a significant increase in the protein expression level and current amplitude ^[65][80]. The expression of the hERG potassium channel, the product of the human ether-a-go-go related-gene, at the membrane of HEK cells, is also regulated by the UPS system ^[66][67][81,82], and proteasomal inhibition by BTZ, MG132, and other drugs rescued trafficking-deficient LQT2-related and schizophrenia-related hERG channel variants ^{[68][69][70][71]}[83,84,85,86]. In addition to calcium and potassium channels, the cardiac Nav1.5 sodium channel is also targeted by the UPS ^[72][87]. MG132 increased its protein expression and current density in isolated neonatal rat cardiomyocytes and rescued the Nav1.5 reduction in cardiomyocytes of dystrophin-deficient mdx5cv mice ^[73][74][88,89]. Interestingly, in Schistosoma mansoni parasites, MG132 caused a decreased expression of transcripts of different ion channels, including the HVA Ca²⁺ channels, Ca²⁺-activated potassium channels, and ATP-sensitive potassium channels, an effect opposite to that observed in different animal models ^[75][90]. That said, it is not surprising that inhibition of the proteasome machinery leads to alterations in excitability, with deleterious effects on neuronal and cardiac activity.

It is well established that chemotherapeutics induce cardiotoxicity to the point that the field of cardio-oncology has developed. Although the definition of cardiotoxicity commonly indicates a decline in patients’ cardiac function, the spectrum of cardiac side effects of chemotherapeutic treatment is heterogeneous and includes impairment in ventricular depolarization or repolarization and QT interval alterations, arrhythmia, bradycardia, tachycardia, decreases in left ventricular ejection fraction and fractional shortening, and irreversible congestive heart failure ^[76][91]. All of which worsen patient quality of life and increase mortality. Anthracyclines are considered the most common culprit drugs causing chemotherapy-induced cardiotoxicity, (acute events in 0.4–41% of patients and chronic events in 0.4–23%) followed by fluoropyrimidines (3–19%) ^[77][78][92,93]. Taxanes are in third position with an epidemiology of 3–20% cardiotoxic events, the most common of which are arrhythmia and cardiac ischemia. In particular, paclitaxel treatment causes acute or sub-acute bradycardia in 30% of patients, cardiac ischemia in 5% of treated patients ^[76][91], heart block, and atrial or ventricular arrhythmias in a smaller fraction of patients (0.5%) and restricted left ventricular pump function, and can provoke chronic cardiotoxicity with clinical symptoms of cardiac insufficiency even decades after the end of treatment ^[79][94].

BTZ and CFZ cardiotoxicity is still a matter of debate. Clinical data are conflicting, as cardiac events are not clearly related to significant cardiovascular risk factors, such as existing cardiac diseases or co-administration of known cardiotoxic drugs ^[47][80][62,95]. Even if rare, BTZ-associated cardiac events have been reported, and include heart failure (the most common), complete atrioventricular block, atrial fibrillation and other forms of arrhythmias, pericardial effusion, orthostatic hypotension, and ischemic heart disease ^[47][55][56][62,70,71]. CFZ is considered more cardiotoxic than BTZ and it has also been associated with a higher incidence of cardiac arrhythmias ^[81][96]. These uncommon events may suggest a mild effect of BTZ and CFZ on the cardiac tissue, that can become life-threatening in the presence of cardiac risk factors and/or compromised substrates.

Animal models have been used to investigate the mechanism behind BTZ and CFZ alleged cardiotoxicity. In male Wistar rats, the administration of BTZ led to left ventricular contractile dysfunction with impaired cardiomyocyte contractility, due to mitochondrial alteration, and reduced ATP production ^[82][97]. Moreover, Hasinoff and colleagues recently tested both BTZ and CFZ on primary neonatal rat cardiomyocytes showing that the two compounds induced cell damage at sub-micromolar concentrations. The study argued that the proteasomal inhibition within a cellular environment, characterized by elevated sarcomeric protein turnover, led to cellular damage and subsequent cell death and apoptosis ^[83][98]. In another study, Tang and colleagues pointed to an overactivation of the hypertrophy-related calcineurin and nuclear factor of activated T-cells (NFAT) signaling pathway as the culprit for BTZ cardiotoxicity in cultured and in vivo murine cardiomyocytes. In particular, the administration of BTZ induced left ventricular hypertrophy, heart failure, and premature death ^[84][99].

Despite the evidence, the mechanisms behind BTZ and CFZ cardiotoxicity remain elusive. As reported above, BTZ has a direct effect on the Cav3.2 level of expression in rat DRGs through inhibition of channel ubiquitination and internalization ^[51][66]. In the heart, the UPS is the principal protein degradation system, managing the turnover of up to 90% of the cellular proteins ^[85][100], and alteration in the UPS can lead to several cardiac diseases, including cardiac hypertrophy, chronic heart failure, and remodeling ^[86][101]. It is, therefore, intriguing that to date no studies have been published regarding the potential cardiomyocyte electrophysiological consequences of inhibition of the cardiac proteasome by BTZ and CFZ and the potential of T-type calcium channels as a pharmacological target. To date, several therapeutic strategies and targets have been proposed to reduce clinical cardiotoxicities, among them iron-chelating drugs, β-blockers, renin-angiotensin-aldosterone system (RAAS) inhibitors, SGLT2 inhibitors, late inward sodium current (INa_L) selective inhibitors, phosphodiesterase-5 inhibitors, metabolic agents, and statins, as well as growth factors and hormones.

Dihydropyridine Ca²⁺-channel blockers (amlodipine, felodipine) have been suggested as first-line agents in the case of fluoropyrimidine treatments, when chemotherapy-induced cardiotoxicities range from QT prolongation to hypertension and left ventricular dysfunction ^[87][102]. Cardiotoxic events were suggested to be mediated by vascular smooth muscle cells and, thus, dihydropyridine Ca²⁺-channel blockers may exert direct vasodilatory effects via the arteriolar smooth muscle. As a side effect, there may be lower extremity edema, the frequency of which is dose-dependent and could be minimized by lowering the dose and by nocturnal administration ^[88][103]. On the other hand, non-dihydropyridine Ca²⁺ channel blockers are not indicated, due to drug-drug interactions and for the impact that they may have on the CYP3A4 system, which can lead to increased concentrations of the chemotherapeutic drug. Arterial hypertension is frequently reported (11–45%) in patients receiving VEGF inhibitors, such as bevacizumab and sunitinib. Ca²⁺ channel blockers are usually prescribed in these cases. In contrast, they should be used with caution in cases of arrhythmias, either supraventricular or ventricular, and in particular in bradyarrhythmias ^[81][96].

A recent review that summarized therapy-specific cardioprotective strategies only mentioned the chemotherapeutic class of proteasome inhibitors, confirming that, even though some information is available on how to treat these cardiotoxicities, robust data on primary cardioprotective strategies are lacking ^[89][104]. Indeed, compared to older classes of anti-cancer agents, proteasome inhibitors have only recently been introduced in clinical practice (the progenitor was approved by the FDA approval in 2008). This could be the main reason why an adequate estimation of their cardiotoxic effects is missing, and why the cellular and molecular mechanisms mediating the cardiotoxicity, and the role of T-type calcium channels, is still poorly explored. It appears clear though, that with the advancement of precision medicine and with the emerging of new classes of chemotherapy and targeted therapy drugs, there is an urgent need to develop novel strategies to mitigate adverse effects and to reduce clinical and subclinical cardiotoxicity.