Targeting calcium regulation for heart failure and arrhythmia therapeutics – a critical review

Abstract

Despite advances in pharmacologic and procedural therapies, heart failure (HF) and cardiac arrhythmias remain significant global health burdens, highlighting the urgent need for novel therapeutic strategies. Defective calcium (Ca²⁺) handling in cardiac myocytes is recognized as a central pathogenic mechanism underlying both HF and atrial and ventricular arrhythmias. In this review, we critically assess the current state of research on Ca²⁺ handling proteins and their role in causing HF and arrhythmias, highlighting therapeutic implications. Recent paradigm-shifting discoveries, clinical trial outcomes, and challenges of targeting Ca²⁺ handling proteins are examined. As outlined in this review, an improved understanding of the relevant proteins and their differential expression and function in human health and disease is crucial for developing Ca²⁺ handling-targeted therapeutics that can fundamentally alter the natural history of HF and arrhythmias.

Introduction

Despite significant progress in pharmacologic and procedural treatment modalities, the burden of cardiovascular disease continues to increase, exacerbated by an aging global population. Heart failure (HF) and both atrial and ventricular arrhythmias are major causes of morbidity and mortality in developed nations. HF is estimated to affect 56 million people worldwide.¹ The prevalence of atrial fibrillation (AF), the most common cardiac arrhythmia, is rising rapidly in the United States from 5.2 million in 2010 to an expected 12.1 million in 2030.² Ventricular arrhythmias, while rarer than atrial arrhythmias, have a high degree of associated mortality; about half of all sudden cardiac deaths (SCD) are attributable to ventricular arrhythmias.³

Current treatment modalities, particularly in HF, tend to target neurohormonal pathways. While these medications improve survival, they do not always halt disease progression—especially in late-stage disease. Despite advances in guideline-directed medical and device therapies, 5-year mortality after an index hospitalization for HF exceeds 50% and recurrent hospitalizations drive enormous healthcare costs.⁴ Similarly, while ICDs and antiarrhythmics reduce SCD, they do not eliminate arrhythmia burden completely and have significant associated morbidity. These realities highlight an urgent unmet need for mechanism-based therapies that go beyond neurohormonal blockade, in the hope that novel therapeutic targets can slow disease progression, reduce mortality, and improve quality of life.

In the heart, appropriate Ca²⁺ handling is essential for normal function. The critical role of Ca²⁺ in cardiomyocyte excitation-contraction coupling (ECC) is well characterized. The pleotropic role of Ca²⁺ in cardiac (patho-)physiology is becoming increasingly evident. Ca²⁺ acts as a key intracellular second messenger, mediating inflammatory pathways, controlling mitochondrial energetics and redox signaling, and playing a role in pathogenic remodeling. Although HF and arrhythmias are complex conditions with many contributing factors, defective Ca²⁺ handling in cardiac myocytes is a central common pathogenic mechanism underlying HF and both atrial and ventricular arrhythmias. As such, considerable recent attention has been devoted to the numerous proteins that regulate Ca²⁺ cycling as potential therapeutic targets.

Here, we review the role of Ca²⁺ in the heart, with a focus on therapeutic targets for HF and cardiac arrhythmias. We start by critically evaluating the major proteins involved in Ca²⁺ handling, highlighting the therapeutic implications of these discoveries. Next, we review both FDA-approved therapies that target Ca²⁺ handling proteins and those previously and currently under clinical investigation. Finally, we discuss challenges in therapeutically targeting Ca²⁺ handling proteins and future directions for research.

A Critical Re-evaluation of the Proteins Underlying Ca²⁺ cycling in Health and Disease

Understanding Ca²⁺ homeostasis in cardiomyocytes requires familiarity with ECC. ECC is the process by which electric excitation of the surface membrane (i.e the action potential) is converted into mechanical force. This process requires highly coordinated Ca²⁺ cycling.

ECC in the cardiomyocyte has five sequential phases resulting in contraction (Fig 1): 1) Initiation and propagation of an action potential along the plasma membrane leading to membrane depolarization; 2) Activation of L-type voltage-gated Ca²⁺ channels (LTCC), Ca_V1.2, leading to rapid influx of Ca²⁺; 3) Ca²⁺ mediated activation of the type 2 ryanodine receptor (RyR2) leading to Ca²⁺ release from the sarcoplasmic reticulum (SR) in a process known as Ca²⁺-induced Ca²⁺ release; 4) Ca²⁺ binding to troponin C causing conformational changes in troponin and subsequently tropomyosin; 5) Actin-myosin crossbridge formation causing sarcomere shortening resulting in muscle contraction.^{5, 6}

Figure 1. Overview of excitation-contraction coupling and calcium cycling proteins in the cardiomyocyte.

Schematic representation of the proteins involved in Ca²⁺ handling in the cardiomyocyte. Targets of the Ca²⁺/Calmodulin kinase signaling pathway are depicted with dotted red arrows. Targets of the protein kinase A signaling pathway are depicted with dotted black arrows. LTCC indicates the L-type Ca²⁺ channel; NCX, sodium-calcium exchanger; β-AR, β-adrenergic receptor; AT₂R, angiotensin 2 receptor; CaM, calmodulin; CaMKII, Ca²⁺/Calmodulin-dependent kinase; cAMP, cyclic AMP; PKA, protein kinase A; PP1, protein phosphatase 1; RyR, ryanodine receptor; CSQ, calsequestrin; SERCA, sarcoendoplasmic reticulum Ca²⁺-ATPase; PLN, phospholamban; SR, sarcoplasmic reticulum; HRC, histidine-rich Ca²⁺ binding protein; S100A1, S100 Ca²⁺ binding protein; Cal2, Calstabin 2. Created in BioRender. Redel-traub, G. (2025)

Relaxation (i.e. diastole) is a similarly tightly regulated process with three primary mechanisms to decrease [Ca²⁺]_cyt occurring in parallel (Fig 1): 1) Ca²⁺ reuptake into the SR via phosphorylation of phospholamban (PLN) and activation of the sarcoendoplasmic reticulum calcium ATPase (SERCA2a); 2) Ca²⁺ transport out of the myocyte primarily via the sodium-calcium exchanger (NCX); 3) Ca²⁺ transport into the mitochondria via the mitochondrial calcium uniporter (MCU). It is important to note that while skeletal and cardiac muscle contraction and relaxation have many similarities, significant differences exist that are beyond the scope of the current review.

Defective ECC leads to cardiomyocyte dysfunction through multiple mechanisms: directly, via impaired contraction and relaxation as well as indirectly via abnormal Ca²⁺ homeostasis and resultant dysregulated energy dynamics, inflammatory responses and pathologic growth signaling.^7–9 As a result, the proteins involved in Ca²⁺ handling have been the subject of extensive research.

Three macromolecular complexes comprising the Ca²⁺ channels Cav1.2, RyR2, and SERCA along with their associated accessory proteins, have been extensively studied. These channels are regulated by several well-studied signaling pathways responsive to changing demands for cardiac output. Abnormalities in the function of proteins within these macromolecular complexes as well as the regulatory signaling proteins, play a central role in the pathogenic mechanisms underlying HF and atrial and ventricular arrhythmias. As such, significant efforts have been made to design therapeutics that target these macromolecular complexes and signaling pathways to treat cardiac pathology.

The LTCC Cav1.2 is the primary entrance pathway for extracellular Ca²⁺ into the cardiomyocyte, enabling rapid Ca²⁺ influx when the plasma membrane is depolarized. Cav1.2 dysfunction is a causative factor in genetic arrhythmias; pathologic mutations in Cav1.2 are associated with short QT syndrome (SQTS), Timothy Syndrome, a variant of long QT syndrome (LQTS), and, disputedly, Brugada Syndrome.^10–12 Cav1.2 is a key target of β-adrenergic regulation in atrial and ventricular cardiomyocytes, with β-adrenergic agonists increasing Ca²⁺ current 2–3 fold.¹³ In HF, a state of chronically high β-adrenergic tone, sustained activation of Cav1.2 leads to changes in expression and distribution along the plasma membrane; in failing human ventricles, the density of Cav1.2 is decreased, but the Ca²⁺ current is maintained via increased channel open probability.^14–16 Older studies suggested that these adrenergically stimulated changes to Cav1.2 play a causal role in hypertrophy, HF, and arrhythmia.^{16, 17}

The β-adrenergic agonist-induced stimulation of Ca²⁺ current is dependent on the phosphorylation of Rad, an inhibitor of Ca²⁺ channels.¹⁸ Rad is a member of the RGK Ras family of proteins that interacts with the Cav1.2 and regulates activation of the channel by PKA.¹⁹ PKA phosphorylation of Rad releases the inhibition of the channel.^{20, 21} By controlling the activity of Cav1.2 channels, Rad is a key effector of inotropy.²² Deletion of Rad, either globally or specifically in the heart, increases basal Ca²⁺ current to levels similar to those induced by adrenergic stimulation. Interestingly, this prolonged increase in Ca²⁺ influx does not lead to hypertrophy, HF, or arrhythmias.^{23, 24} This suggests that adrenergically stimulated changes to Ca_V1.2 (via Rad phosphorylation) are insufficient to induce cardiac remodeling and HF; other downstream targets of PKA, such as those discussed below, must be responsible for and/or synergize with increased Ca²⁺ current to result in pathological remodeling.

The macromolecular Cav1.2 complex is localized to transverse tubule (T-tubule) membrane invaginations forming associated cardiac dyads with the RyR2 macromolecular complex. Recent discoveries over the last decade have helped elucidate the organization of T-tubules and their importance in regulating intracellular Ca²⁺ transients in normal and pathologic states. T-tubule remodeling is an early change in HF; junctophilin-2 (JPH2) and bridging integrator 1 (BIN1) are the most well-characterized T-tubule structural proteins differentially expressed in HF. ^25–27 There have been mixed results on the overexpression of JPH2 in HF models. It remains unclear whether reductions in JPH2 are the result of HF progression or a cause. ²⁸ More recent studies, have implicated JPH2 cleavage by calpain-2 as the key mediator in the disruption of the T-tubule; it is possible that targeting calpain-2 induced cleavage may be a novel therapeutic strategy for HF.^{29, 30} BIN1 overexpression, has also been shown to restore T-tubule structure and rescue LV function in large animal models of failing hearts, making it an appealing potential therapeutic, which will be tested in future human clinical trials.³¹

RyR2 is found on the SR membrane of cardiomyocytes and forms the other half of the cardiac dyad, existing in close apposition to Cav1.2. RyRs are homotetrameric intracellular Ca²⁺ release channels comprising four 565 kDa (^~5,000 amino acids) protomers. It is now generally accepted that defective SR Ca²⁺ handling in cardiomyocytes plays a crucial role in the pathophysiology of HF and arrhythmias. This defective SR Ca²⁺ handling is predominantly characterized by leaky RyR2 channels resulting from stress-induced dissociation of the RyR2 stabilizing subunit calstabin2 (also known as FKBP12.6).^{32, 33} Chronic diastolic SR Ca²⁺ leak due to stress-induced remodeling—caused by PKA phosphorylation and oxidative stress leading to other post-translational modifications to RyR—depletes SR Ca²⁺, reduces contractility, promotes HF progression, and can generate arrhythmogenic delayed afterdepolarizations (DADs).^34–36 While targeting calstabin2 has proved a promising clinical target, therapeutics targeting oxidative stress in clinical trials have been less successful perhaps due to the newly discovered redox-sensitive binding of RyR with ERp44—a protein that binds and stabilizes RyR under oxidative conditions. ³⁶

Many germline RyR2 mutations cause SR Ca²⁺ leak and are considered pathogenic. RyR2 mutations cause most cases of catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited disorder characterized by ventricular arrhythmias during exercise in a structurally normal heart.^{37, 38} Given the central role leaky RyR2 plays in HF and arrhythmias, significant research has focused on stabilizing RyR2, leading to active clinical trials.³⁹ In addition to mutations directly affecting RyR2, pathogenic mutations of calmodulin (CaM)—a key regulatory protein of both RyR2 and LTCC that helps control channel dynamics—have been shown to cause inherited arrythmia syndromes including CPVT, LQTS and idiopathic VF. These calmodulinopathies often present with severe, early-onset phenotypes and carry a high risk of SCD, underscoring the urgent need for targeted interventions. To date, however, no effective treatments have been developed specifically for calmodulinopathies.⁴⁰

SERCA2a is the primary protein responsible for Ca²⁺ reuptake into the SR during diastole. As discussed above, decreased SR Ca²⁺ content is a defining feature of failing hearts. Decreased SR Ca²⁺ is primarily driven by an increased SR Ca²⁺ leak via RyR2 and decreased reuptake by SERCA2a into the SR. Thus, SERCA2a has long been posited as central in the cellular pathophysiology of HF. Long-standing dogma in the field holds that SERCA2a expression is reduced at the transcript and protein levels in failing hearts.^{41, 42} Reduced expression combined with increased inhibition by PLN, an inhibitor of SERCA2a, has been proposed to contribute to HF.⁴³ Early preclinical models of HF appeared to corroborate this: cardiomyocyte-specific deletion of SERCA2a caused HF under basal conditions and gene transfer of SERCA rescued cardiac function in various large animal models of HF.^{44, 45} However, recent data have called this dogma into question. In the largest proteomic analysis of cardiac tissue from patients with end-stage HF, targeted and untargeted proteomics revealed no difference in SERCA2a protein abundance compared to control hearts.⁴⁶ Unaltered protein expression does not necessarily rule out impaired SERCA2a activity; post-translational modifications to SERCA2a in HF have been shown to alter its function.⁴⁷ However, these recent proteomics findings do call into question a major tenet of molecular HF pathophysiology that has received much notice in the field over the past two decades and now must be rethought.⁴⁶

In its unphosphorylated form, PLN inhibits SERCA2a Ca²⁺-affinity, and phosphorylation by Protein Kinase A (PKA) and Ca²⁺/Calmodulin kinase (CaMKII) relieves this inhibition and enhances reuptake of Ca²⁺ in the SR. Most studies report unchanged protein levels of PLN and decreased phosphorylation of PLN in failing hearts.⁴⁶ Decreased PLN phosphorylation in HF leads to increased PLN bound to SERCA2a and depressed SR Ca²⁺ concentration—a hallmark of HF, as mentioned above. ^{46, 48} Germline mutations in PLN, leading to a super-inhibitory form or abnormal interaction with PKA, have been linked to hypertrophic and dilated cardiomyopathy.^{49, 50} A genetic mutation in PLN (R14del) has also been linked to arrhythmogenic cardiomyopathy in humans.⁵¹ Various pre-clinical therapeutics targeting PLN using antisense oligonucleotides, intrabodies, and AAVs have successfully restored cardiac function in HF models.^52–54 There are ongoing clinical trials targeting PLN, which are discussed below.

Protein phosphatase 1 (PP1) is a serine-threonine phosphatase localized to the SR membrane that regulates cardiac contractile performance in conjunction with its inhibitor I-1c.⁵⁵ PP1 and I-1c have been implicated as fine-tuning regulators of β -adrenergic stimulation in the heart.⁵⁶ PP1, a phosphatase of PLN, maintains PLN-mediated SERCA inhibition. In response to β-AR agonism, PKA phosphorylates and thereby activates I-1c, inhibiting PP1, allowing for increased PLN phosphorylation and SERCA-mediated SR Ca²⁺ reuptake. In HF, there is reduced I-1c expression and activity as well as increased PP1 expression and activity, which synergize with changes in PLN described above to reduce SERCA-mediated SR Ca²⁺ reuptake.^{57, 58} Augmenting I-1c activity has been shown to restore function in HF models.⁵⁹ Moreover, genetic delivery of active I-1c did not significantly increase arrhythmias, which was a concern due to PP1/I-1c’s role in regulating RyR. Successful pre-clinical data have led to human trials on I-1c activation via gene therapy.

Several accessory proteins in the SR are associated with both RyR and SERCA2a, forming a part of both respective macromolecular complexes. Calsequestrin (CSQ) is a high-capacity Ca²⁺ binding protein in the SR lumen. CSQ is linked to the SR membrane via junctin and triadin, forming a macromolecular complex that modulates RyR Ca²⁺ release and SERCA2a reuptake.⁶⁰ Alterations in CSQ, junction, and triadin levels are associated with pathologic remodeling and ventricular arrhythmias. Loss-of-function CSQ mutations have been shown to cause an autosomal-recessive form of CPVT.⁶¹ Junctin levels are significantly downregulated in HF and nearly undetectable in human failing hearts.⁶² Decreased junctin worsens SR Ca²⁺ leak and contributes to susceptibility to arrhythmias in HF.⁶³

The histidine-rich Ca²⁺ binding protein (HRC), like CSQ, is a low-affinity, high-capacity Ca²⁺ binding protein in the SR. HRC is linked primarily to the plasma membrane via triadin and modulates Ca²⁺ release from and reuptake into the SR via its interaction with RyR and SERCA.⁶⁴ HRC is significantly down-regulated in patients with HF. ⁶⁵ A genetic variant of HRC, Ser96Ala, which abolishes a key phosphorylation site, is associated with ventricular arrhythmias in patients with idiopathic dilated cardiomyopathy.⁶⁶ HRC mutations have also been associated with Progressive familial heart block type I and Isolated cardiac conduction disease.^{67, 68} Despite their apparent importance in regulating SR Ca²⁺ release and reuptake, there are currently no CSQ-, junctin-, triadin- or HRC-related therapies in human trials.

S100 Ca²⁺ binding protein A1 (S100A1) is yet another low-affinity, high-capacity Ca²⁺ binding protein with expression in the SR, mitochondria, and myofilaments; its primary role in Ca²⁺ handling is likely through modulation of RyR and SERCA activity.^{69, 70} S100A1 protein levels are reduced in human failing hearts, and higher reductions in S100A1 are associated with worse contractile function.^{71, 72} Numerous studies have shown that overexpression of S100A1 leads to increased Ca²⁺ transients and improved contraction. Moreover, S100A1 gene transfer in both small and large animal HF models rescues contractile performance and improves mortality.^73–75 A clinical trial of a triple gene plasmid construct including constitutively active S100A1, stromal cell-derived factor-1α (SDF-1α), and vascular endothelial growth factor 165 (VEGF165) has recently completed a phase 1 trial in LVAD patients.⁷⁶ Gene therapy targeting S100A1 alone has not yet been developed for use in clinical trials.

NCX is the major Ca²⁺ efflux mechanism in the cardiomyocyte and, therefore, plays a critical role in diastolic relaxation and the regulation of intracellular Ca²⁺ content. In the failing heart, increased expression and activity of NCX contribute to the depletion of SR Ca²⁺, which is associated with contractile dysfunction.^{77, 78} Cardiac glycosides, such as digitalis, have been used to indirectly affect NCX activity in the failing heart.⁷⁹ By inhibiting the Na⁺/K⁺ ATPase, leading to increased intracellular Na⁺, digitalis reduces ‘forward’ NCX activity and can even reverse the exchanger leading to Ca²⁺ influx. These changes ultimately favor the accumulation of Ca²⁺ in the SR, improving contractility.^{80, 81} Increased NCX expression and activity have also been implicated in atrial and ventricular arrhythmias by prolonging action potentials and promoting DADs.^{82, 83} Given the role of increased expression and function of NCX in both HF and arrhythmia, selective inhibition of NCX has long been viewed as an attractive therapeutic option. There have been mixed results in HF and arrhythmia models in pre-clinical trials of several potential NCX blockers.^84–86 One non-selective NCX inhibitor is undergoing an early-phase clinical trial, which will be discussed below.

As previously alluded to, the proteins involved in ECC are regulated by multiple signaling pathways that respond to changing demands for cardiac output. Of particular importance are PKA and CaMKII. PKA is a serine-threonine kinase that is a central regulator of cardiac performance, modulating ECC, growth, metabolism, and energy production.⁸⁷ The canonical PKA signaling pathway is essential for adrenergic stimulation to the heart. In response to adrenergic stimulation, increases in cAMP activate PKA, which then targets many of the essential proteins in ECC and Ca²⁺-handling discussed in this review, including Rad, PLN, RyR, troponin-I, and myosin binding protein-C. PKA thereby exhibits crucial effects on inotropy, chronotropy, and lusitropy by increasing Cav1.2 Ca²⁺ influx, increasing SR Ca²⁺ release and reuptake, and modulating myosin Ca²⁺ sensitivity.⁸⁸ In HF, a state of chronically elevated adrenergic tone, PKA activity becomes dysregulated, leading to abnormal gene expression, impaired energy production and metabolism, and abnormal Ca²⁺ handling.⁸⁷ Chronic overactivation of PKA in failing hearts results in increased phosphorylation of RyR, leaky RyR, and decreased SR Ca²⁺ content.⁸⁹ Direct PKA inhibitors are not of benefit for HF and arrhythmias, likely due to the pleiotropic role of PKA.

CaMKII is a serine-threonine kinase that is abundant in the myocardium and activated by increased intracellular Ca²⁺ and ROS and adrenergic stimulation. CaMKII phosphorylates a diverse array of proteins involved in ECC and cell death and promotes transcriptional activation of inflammation and hypertrophy. Increased transcription, expression, and activity of CaMKII potentially affect downstream processes that contribute to HF and atrial and ventricular arrhythmias.⁹⁰ CaMKII is activated by the β-adrenergic receptor, angiotensin II receptor, and aldosterone receptor, three validated therapeutic targets for HF and arrhythmias. It has been proposed that at least part of the therapeutic benefit of β-blockers, angiotensin receptor blockers, and mineralocorticoid receptor antagonists is due to decreased CaMKII activity, highlighting the potential therapeutic value of direct CaMKII modulation.^91–93 In HF and arrhythmia models, CaMKII inhibition is protective against HF and arrhythmias under various stressors.^{92, 94, 95} Several CaMKII inhibitors have undergone clinical trials for both HF and arrhythmias, discussed below.

Current Therapeutics

Over the last 70 years, significant advancements have been made in the treatment of HF and cardiac arrhythmias. Guideline-directed medical therapies generally target either the renin-angiotensin-aldosterone system (RAAS) or the β-adrenergic pathways and, more recently, metabolic pathways. While this review focuses on the novel therapeutic approaches arising from research on Ca²⁺ handling proteins, it is worthwhile to briefly discuss currently approved pharmacological therapies, including their impact on Ca²⁺ handling in the heart (Fig 2).

Figure 2. Therapeutic targeting of calcium handling proteins for heart failure and arrhythmias.

Schematic representation highlighting key proteins involved in Ca²⁺ cycling cardiomyocyte that have emerged as therapeutic targets. Both investigational agents recently or currently in clinical trials as well as FDA-approved agents are shown in relation to their specific molecular targets. LTCC indicates the L-type Ca²⁺ channel; NCX, sodium-calcium exchanger; β-AR, β-adrenergic receptor; AT₂R, angiotensin 2 receptor; ACE, angiotensin coverting enzyme inhibitor; ARB, angiotensin receptor blocker; ARNI, angiotensin receptor/neprolysin inhibitor; MR, mineralocorticoid receptor; MRA mineralocorticoid receptor antagonist; CaMKII, Ca²⁺/Calmodulin-dependent kinase; PP1, protein phosphatase 1; PLN, phospholamban; RyR, ryanodine receptor; SR, sarcoplasmic reticulum; S100A1, S100 Ca²⁺ binding protein; Cal2, Calstabin 2. Created in BioRender. Redel-traub, G. (2025)

HF and many cardiac arrhythmias are characterized by their hyper-adrenergic tone. While the initial elevation in sympathetic nervous system activation is beneficial by maintaining contractility, over time, chronic activation of β -adrenergic receptors promotes HF and arrhythmias.⁸⁸ Therapeutic targeting of excessive β-adrenergic activation is achieved through the use of β-AR blockers. Carvedilol, bisoprolol and sustained release metoprolol carry a Class I recommendation in HFrEF, reducing all-cause mortality by 31% and HF hospitalizations by 25% in meta-analyses. ^{4, 96} Some of the beneficial effects of β-blockers are likely due to reduced PKA-mediated phosphorylation of RyR2, resulting in stabilization of leaky RyR2 channels. Indeed, by preventing PKA phosphorylation of the channel and the resulting dissociation of calstabin2, which promotes diastolic SR Ca²⁺ leak in failing hearts, β-blockers can improve contractility and reduce arrhythmias in failing hearts.^{97, 98}

RAAS signaling, which is elevated in HF, is proarrhythmic, partly due to direct effects on ion channels, including increased Ca²⁺ current, and partly due to the formation of a proarrhythmic substrate from pathologic remodeling and HF.^{1, 99} Several therapeutics target the RAAS system for patients with HF: angiotensin converting enzyme inhibitors, angiotensin receptor blockers, angiotensin-receptor-neprilysin inhibitors, and mineralocorticoid receptor antagonists all carry a Class I recommendation in HFrEF, and have been shown to reduce HF hospitalizations by 20–30% and all-cause mortality by 15–27% according to meta-analyses. ^{4, 100, 101} Although not formally anti-arrhythmic, RAAS inhibitors reduce SCD and atrial arrhythmias in patients taking these medications.^{102, 103} While RAAS inhibition has pleiotropic benefits in HF and arrhythmia, at least some of the benefits may be related to improved Ca²⁺ handling in cardiomyocytes.^{99, 104, 105}

Two types of Ca²⁺-channel blockers, non-dihydropyridines and dihydropyridines, directly affect ECC and Ca²⁺ handling. Dihydropyridines (e.g., amlodipine, nifedipine) are more vascular selective, while non-dihydropyridines (e.g., diltiazem, verapamil) are more myocardial selective. Ca²⁺-channel blockers, except for amlodipine, carry a Class III (harm) recommendation in HFrEF, due to their negative inotropic effect which worsens survival in late stage disease.⁴ Ca²⁺-channel blockers remain useful for atrial and select ventricular arrhythmias, but their use must be balanced against LV function.²

Calcium Handling Proteins are Novel Therapeutic Targets:

The AHA/ACC/HFSA guidelines recommend β-blockers and RAAS-inhibitors as first-line therapies to improve morbidity and mortality in HF.⁴ However, not all HFrEF patients are on guideline directed medical therapy and even fewer are prescribed optimal doses, in part due to the effects of these medications outside of the myocardium such as hypotension and renal injury. As previously mentioned, there is significant interest in directly targeting Ca²⁺ handling proteins to alter the disease course of HF and arrhythmia, thereby reducing morbidity and mortality. Decades of research have led to clinical trials on therapeutics directly targeting Ca²⁺ handling proteins. Here, we will detail the therapeutics that have been studied in human trials. (Fig 2, Table 1).

Table 1. Summary of therapeutics targeting cardiac calcium handling proteins in clinical trials.

Comprehensive overview of clinical-stage therapeutics designed to modulate cardiac calcium handling for heart failure and arrhythmias. Listed are therapeutic agents, their molecular targets, proposed mechanisms of action, clinical trial status (Phase 1, 2, or 3), the results of completed trials, and relevant patents. This table summarizes both the successes and challenges encountered in clinical translation, emphasizing areas of continued investigation and potential clinical impact. Up to date as of June 2025.

Target	Drug	Mechanism	Trial ID	Trial Details/Results (if available)	Patent Applications
SERCA2a	AAV1/SERCA2a	Gene transfer of SERCA2a	Phase 2: CUPID 1, CUPID 2, AGENT-HF, SERCA-LVAD, MUSIC-HFpEF, MUSIC HFrEF	No improvement in outcomes. MUSIC trials currently enrolling	US 8,122,122 B2 (Methods for treating HF by delivering SERCA2a)
	Istaroxime	Blocks SERCA2a-PLN Interaction	Phase 2: SEISMiC, Horizon-HF	Hemodynamic, echocardiographic improvement in patients hospitalized with HF	US 11,197,869 B2 (Intravenous istaroxime for acute HF)
	Neuregulin-1 (NRG-1)	Growth factor activating ERBb/HER2 pathway preventing changes to expression of SERCA2a, NCX, LTCC	Phase 2: NCT01251406 Phase 3: NCT03388593	Phase 2: Hemodynamic, echocardiographic improvement in patients with HF Phase 3: Actively enrolling	US 11,246,909 B2 (Neuregulin based methods for treating HF)
	JK-07	Human monoclonal antibody containing an active peptide fragment of NRG-1. Otherwise same as above	Phase 2: RENEU-HF (NCT06369298)	Actively enrolling	US 11,242,370 B2 (Neuregulin compounds and methods of use)
PLN	AB-1002 (NAN-101)	Gene transfer of constitutively active I-1c	Phase 2: GenePHIT (NCT05598333)	Actively enrolling	WO 2022/031914 A2 (AAV vectors for treatment of HF
	RT-100 (Ad5.hAC6)	Gene transfer of AC6➔downregulation of PLN	Phase 2: NCT00787059 Phase 3: FLOURISH (NCT03360448)	Phase 2: Hemodynamic, echocardiographic improvement in patients with HF Phase 3: Enrollment planned for 2025	WO 2001/048164 A2 (AC6 therapy for treatment of HF)
	Cimlanod (BMS-986231)	Nitroxyl modifying cysteine residues on PLN/SERCA	Phase 2: STAND-UP AHF (NCT03016325), NCT03730961	Transient improvement in markers of congestion during infusion, which do not persist	US 20120201907 A1 (Nitroxyl progenitors in the treatment of HF)
RYR2	JTV-519	Stabilization of leaky RyR2	Phase 2: NCT00626652	N/A	US 7,879,840 B2 (Agents for treating disorders involving the ryanodine receptor)
	ARM210	Stabilization of leaky RyR2	Phase 2: NCT05122975	Enrolled patients with CPVT, enrollment complete, results pending	US 7,879,840 B2 (Agents for treating disorders involving the ryanodine receptor)
	Flecainide	Stabilization of leaky RyR2	NCT01117454	In patients with CPVT, Flecainide plus β -blockers reduced ventricular ectopy during exercise compared with β-blocker alone.	N/A
	Dantrolene	RyR antagonist	Phase 3: NCT04134845	Enrolled patients with structural heart disease planned for VT ablation, results pending	US 11,123,328 B2 (Dantrolene for treatment of arrhythmias)
CaMKII	CRD-4730	CaMKII inhibitor	Phase 2: NCT06005428	Actively enrolling patients with CPVT	WO 2016/037106 A1 (CaMKII inhibitors and uses thereof)
	NP202	CaMKII inhibitor	Phase 2: NCT02557217	No improvement in LV remodelling for patient’s with anterior STEMIs	WO 2016/037106 A1 (CaMKII inhibitors and uses thereof)
S100A1	INXN-4001	Triple gene plasmid with constitutively active S100A1, SDF-1α, VEGF165	Phase 1: NCT03409627	Safe and well-tolerated	WO 2017/083750 A1 (Polynucleotides encoding S100A1

Targeting SERCA/PLN

Modulating Ca²⁺ reuptake into the SR has been identified as a promising therapeutic target for HF. Successful pre-clinical experiments that rescued large animal models of HF by targeting proteins in the SERCA2a-PLN complex have supported this, and several of these therapies have since progressed to clinical trials.^{44, 45}

The largest clinical trials on SERCA2a modulation were the Phase1/2 CUPID1 and CUPID2 trials, which occurred in the 2010s.^{106, 107} These trials tested a recombinant AAV serotype 1 (AAV1) expressing the SERCA2a gene as a method for gene transfer therapy in HF patients. The initial 39-patient, randomized, double-blind, placebo-controlled CUPID1 trial showed that a single AAV1/SERCA2a intracoronary infusion was safe and feasible and reduced the number of cardiac events as compared to placebo at 1 year. Unfortunately, the larger follow-up CUPID2 trial did not confirm the pilot studies. Two other smaller clinical trials, AGENT-HF and SERCA-LVAD, using an AAV1/SERCA2a strategy in patients with HF and LVAD, respectively, were stopped prematurely when CUPID2 showed neutral results.^{108, 109}

Several theories have been posited to explain the neutral results in CUPID2. Those who support SERCA2a as a viable target argue that the neutral results may stem from ineffective delivery of the vector to the myocardium. To this point, two ongoing trials of patients with HFrEF and HFpEF have implemented an increased therapeutic dose of AAV1/SERCA2a.^{110, 111} Another possibility, supported by a paradigm-shifting recent study described above, is that SERCA2a levels are not reduced in HF. ⁴⁶ While unaltered protein levels do not imply unaltered SERCA2a activity, these findings using state-of-the-art proteomics, in combination with the failure of SERCA2a overexpression to show clinical benefit in human trials, suggest that SERCA2a overexpression may not be an appealing target for therapy. At the least, it appears prudent to utilitze state-of-the-art proteomic data to guide target selection for future clinical trials, especially for therapeutics whose mechanism of action is overexpression via viral gene transfer.

Other therapeutics targeting SERCA2a have also reached clinical trials. Istaroxime, an androstenedione derivative, has lusitropic and inotropic effects on the heart via dual mechanisms of action: 1) inhibition of the Na⁺/K⁺ ATPase and 2) activation of SERCA2a by modulation of the SERCA2a-PLN interaction.¹¹² Initial randomized, placebo-controlled trials on infusion of Istaroxime in the late 2000s included patients hospitalized with acute decompensated HF and showed improved hemodynamic and echocardiographic parameters.^{113, 114} More recent trials have also demonstrated the hemodynamic and echocardiographic benefits of Istaroxime in hospitalized patients with cardiogenic shock.^{115, 116} Phase 3 trials of Istaroxime are forthcoming.

Another therapeutic that works, at least partially through SERCA2a, is neuregulin-1 (NRG-1). NRG-1 is a growth factor that activates the ERBb/HER pathway and is upregulated in response to cardiac stress and subsequently downregulated in late-stage HF. It is posited that the HER/ERBb monoclonal antibody trastuzumab’s cardiotoxicity is primarily mediated by affecting NRG/ERBb signaling.¹¹⁷ While the mechanism of the benefit of NRG-1 for HF is not fully understood, delivery of recombinant NRG-1 increases Cav1.2 density and SERCA2a expression and decreases NCX expression in a rat model of HF.¹¹⁸ Phase 2 trials have demonstrated the safety, tolerability, and efficacy of NRG-1 in patients with HF.^{117, 119} A phase 3 trial is actively enrolling. Participants for a phase 2 trial studying JK-07, a human monoclonal antibody containing an active peptide fragment of NRG-1, are being recruited. ¹²⁰

Modulation of PLN activity, as another route to affect SR Ca²⁺ reuptake, has also been a fruitful avenue of therapeutic discovery, with several therapeutics targeting PLN progressing to clinical trials. AB-1002 (also known as NAN-101) is a cardiotropic AAV that delivers a constitutively active I-1c to cardiomyocytes via intracoronary infusion; this treatment was safe and showed promising results in a Phase 1 trial.¹²¹ Constitutively active I-1c exerts its effect by increasing the phosphorylation of PLN, thereby decreasing SERCA2a inhibition. A phase 2 trial of AB-1002 is actively enrolling patients.¹²²

Gene therapy has also been used to overexpress adenylyl cyclase 6 (AC6)—a protein that primarily catalyzes the conversion of ATP to cAMP but also has been shown to downregulate expression of PLN independent of cAMP concentration.¹²³ In a phase 1/2 randomized, placebo-controlled clinical trial, intracoronary delivery of RT-100 (also known as Ad5.hAC6) was safe, increased LV function, and trended towards reducing cardiac events.¹²⁴ Based on these promising results, a phase 3 trial, FLOURISH, was initially planned in 2018 but was subsequently withdrawn before recruitment due to a change in development plans.¹²⁵ The developer of RT-100 is planning to begin a phase 3 trial shortly.

Cimlanod (also known as BMS-986231) is a second-generation nitroxyl that modifies cysteine residues on PLN, SERCA2a, and RyR, thereby enhancing Ca²⁺ cycling via reduced PLN-mediated SERCA2a inhibition.¹²⁶ Initial phase 1 and 2 trials demonstrated the safety of intravenous infusion of Cimlanod and suggested hemodynamic benefits in patients with acute decompensated HF.^{127, 128} Three additional randomized, placebo-controlled trials have recently shown that infusion of Cimlanod only transiently reduces markers of congestion in acute HF while the drug is being infused and that the drug blunts diuretic response likely due to vasodilation.^129–131 No additional trials of Cimlanod have been announced.

Targeting the Ryanodine Receptor/Calcium Release Channel

With the recent discovery of the central role leaky RyR2 plays in HF and arrhythmias, multiple therapies attempting to stabilize RyR2 have now undergone clinical trials. A phase 2 clinical trial evaluating the effects of JTV-519, a benzodiazepine derivative, on sinus rhythm restoration in patients with AF was conducted, though no data are available.¹³² Rycals are a newer derivative of JTV-519 that bind to and stabilize RyR2 channels by preventing dissociation of the stabilizing subunit calstabin2, thereby reducing Ca²⁺ leak from the SR. Rycals are orally available small molecules that bind directly to RyR1 (skeletal muscle) and RyR2 (cardiac muscle) and fix the channel leak by allosterically stabilizing the closed state of the channel.^{133, 134} The Rycal drug binding site has been identified in RyR1 and RyR2 using cryogenic electron microscopy, showing that the drug binds near the periphery of the cytosolic shell of the channel, far away from the channel pore.^133–135 By stabilizing domain interactions in the channel, Rycal drug binding can stabilize the closed state of the pore and prevent pathological leak of Ca²⁺.¹³³ Importantly, Rycals do not block the channel and are not inotropes. They improve contractility by reducing diastolic SR leak.^{134, 135} This enables restoration of SR Ca²⁺ content, thereby increasing the Ca²⁺ transient and strengthening muscle contraction.^{33, 136}

After animal research showed the benefits of Rycals in models of ischemic HF, AF, and CPVT, multiple clinical trials were initiated.^{137, 138} A clinical trial using a first-generation Rycal, ARM036, in patients with chronic HFrEF showed a reduction in BNP levels and LV systolic and diastolic volumes after 12 weeks of treatment. Currently, a Rycal, ARM210, is undergoing a phase 2 clinical trial for patients with CPVT.¹³⁹ A clinical trial in patients with RyR1-related myopathy using ARM210 showed improved muscle strength and significant improvement in patient-reported fatigue.¹⁴⁰ Thus, RyR-targeted therapeutics can uniquely treat skeletal and cardiac muscle dysfunction.

In addition to experimental agents, flecainide, a class IC antiarrhythmic, has shown efficacy in reducing arrhythmias in CPVT possibly by directly reducing RyR2-mediated SR Ca²⁺ leak, in addition to its sodium channel-blocking effects. ¹⁴¹ Current guidelines recommend considering the use of flecainide in patients for whom arrhythmias persist despite treatment with a β -blocker.^{142, 143}

Finally, dantrolene, a RyR-targeted therapeutic that is used as a skeletal muscle relaxant and to prevent malignant hyperthermia due to inhaled anesthetics during surgery, is currently being tested in patients with HF and ventricular arrhythmias.^{144, 145}

Targeting CaMKII

CaMKII, which can become constitutively active in HF and AF, has been another major focus of drug development for HF and arrhythmia.⁹⁰ NP202, an orally active inhibitor of CaMKIIδ, reduced infarct size and prevented progression to HF in animal models.¹⁴⁶ On this basis, a large phase 2 randomized, placebo-controlled clinical trial was conducted to test the safety and efficacy of NP202 in patients with anterior ST elevation myocardial infarction (STEMI). Although the drug was well tolerated, it did not have any effect on LV remodeling post-MI.¹⁴⁷ Interestingly, two PKC-δ inhibitors, KAI-9803 and Delcasertib also showed promise in reducing infarct size in animal models of MI, but showed no effect on adverse cardiac remodeling in phase 2 trials in patients with anterior STEMIs.^{148, 149}

Despite the negative results for NP202 after anterior STEMI, CaMKII inhibition is still considered a potential target for HF and arrhythmias. Another orally active CaMKII inhibitor, CRD-4730, is undergoing a phase 2 trial for patients with CPVT.¹⁵⁰ Despite the recent interest in CaMKII (and PKC-δ) as potential therapeutic targets for HF and arrhythmia, the exact mechanism of benefit of CaMKII inhibition is not well understood. This is primarily because CaMKII has so many substrates, making it challenging to identify a single or even a few such substrates that promote HF and arrhythmia. Further complicating CaMKII as a therapeutic target is that multiple isoforms and splice variants exist, sometimes redundantly, in the cardiomyocyte such that targeted inhibition may be compensated.¹⁵¹ Further research into the signaling pathway and how individual isoforms and splice variants operate in health and disease may reveal a more precise therapeutic target for future drug design.

Challenges and Future Directions

This review identifies potential therapeutic targets in the key pathways regulating Ca²⁺ handling in cardiac muscle. Over the last decades, great strides have been made in understanding Ca²⁺ handling proteins as a potential cause of, and therapeutic target for, HF and arrhythmias at the cellular, animal, and human genetic levels. These efforts have led to multiple clinical trials, though a blockbuster therapeutic affecting these pathways remains elusive. How do we explain the lack of a major therapeutic targeting Ca²⁺ handling proteins for human HF and arrhythmias? One possible explanation is that our pre-clinical paradigms regarding the role of these proteins in HF and arrhythmia do not extend to human disease. As previously alluded, there is controversy regarding SERCA2a expression and leaky RyR2’s role in human HF and arrhythmia.^{46, 152} Another potential explanation is that Ca²⁺ handling proteins are hard to target because of the pleiotropic role of Ca²⁺ in cellular physiology. As mentioned throughout this review, Ca²⁺ is involved in many roles in the cardiomyocyte including ECC, inflammatory and hypertrophic signaling, mitochondrial energetics, development and cell differentiation, and apoptosis to name just a few. The disrupted Ca²⁺ homeostasis in HF and arrhythmia, therefore, causes abnormalities in many of these Ca²⁺ dependent pathways; a successful Ca²⁺ therapeutic for cardiac pathology will therefore need to not only address abnormalities in ECC, but also reverse (or slow) these other Ca²⁺ dependent abnormalities. Still, targeting Ca²⁺ handling remains a mechanistically appealing focal point to develop novel therapeutics for HF and arrhythmias. With an improved understanding of the relevant proteins and their differential expression and function in human health and disease, as outlined in this review, we remain optimistic that future Ca²⁺ handling targeted therapeutics are on the horizon.

Non-standard Abbreviations and Acronyms

HF

Heart Failure

Ca²⁺

Calcium

SCD

Sudden Cardiac Death

AF

Atrial Fibrillation

ECC

Excitation-Contraction Coupling

LTCC

L-type Voltage-Gated Ca²⁺ Channels

RyR

Ryanodine Receptor

SR

Sarcoplasmic Reticulum

PLN

Phospholamban

SERCA

Sarcoendoplasmic Reticulum Calcium ATPase

NCX

Sodium-Calcium Exchanger

LQTS

Long QT Syndrome

T-Tubule

Transverse Tubule

JPH2

Junctophilin-2 (JPH2)

BIN1

Bridging Integrator 1

DADs

Delayed Afterdepolarizations

CPVT

Catecholaminergic Polymorphic Ventricular Tachycardia

PKA

Protein Kinase A

CaMKII

Ca²⁺/Calmodulin kinase

PP1

Protein Phosphatase 1

CSQ

Calsequestrin

HRC

Histidine-Rich Ca²⁺ Binding Protein

S100A1

S100 Ca²⁺ Binding Protein A1

RAAS

Renin-Angiotensin-Aldosterone System

NRG-1

Neuregulin-1

STEMI

ST Elevation Myocardial Infarction