TnI has evolved into three isoforms in higher vertebrates, encoded by three related genes: TNNI1, TNNI2 and TNNI3. Cardiac TnI (cTnI) in the adult is specifically expressed and regulated by TNNI3, slow and fast skeletal muscle cells by TNNI1 and TNNI2, respectively [83]. In the human chromosomal genome, TNNI3 is located at 19q13.4, encoding approximately 210 amino acids residues with a molecular weight of 24.0 kDa [84]. The functional domain in cTnI between residues from 61 to 112 binds TnT. The inhibitory domain, including residues from 147 to 163, bind strongly to actin and the N-terminal of TnC. It is necessary for regulating the connection of Ca${}^{2+}$ to TnC and actomyosin ATPase activity [85, 86]. A second actin-binding site, residues 168 to 188 of cTnI, binds specifically to the actin-tropomyosin filament contributing to the inhibitory activity of cTnI [87]. The C-terminal domain in cTnI is specific and crucial for normal cardiac relaxation. In addition, the remaining C-terminal part residues from 192 to 210 are not fully identified. Still, they are suspected of playing a significant role in stabilizing the Ca${}^{2+}$-activated state of tropomyosin in the actin filaments [88].

Most RCM-associated mutations in TNNI3 are generally missense rather than frameshift or splice mutations. It has been described that a c.87A$>$G nucleotide substitution in exon 8 of TNNI3 identified by linkage analysis and direct gene sequencing was highly correlated with marked restrictive filling and a family history of sudden cardiac death [17]. The index case of familial occurrence with RCM involved a proband who suffered from severe heart failure at the age of 11. Subsequent investigation in this study revealed that six missense variants were associated with RCM-related specific genetic mutations: p.D190H, p.R192H, p.K178E, p.R145W, p.A171T and p.L144Q [17]. These mutations largely increase the myofibril sensitivity to Ca${}^{2+}$ and affect the basal and maximal actomyosin ATPase activity [89, 90]. The worst clinical phenotypes involved p.K178E and p.R192H resulting in significant increases in Ca${}^{2+}$ sensitivity [90]. According to the echocardiography results of a p.R193H transgenic mouse model, there were significantly reduced left ventricular end-diastolic volumes compared with the wild-type group [91]. Moreover, p.R193H mutant of TNNI3 in adult rat cardiac myocytes further dissected that the increased basal mechanical force cannot be explained by a gain of myofibril Ca${}^{2+}$ sensitivity. It was inferred that the TnI-based disinhibition in actin-myosin interaction at normal diastolic Ca${}^{2+}$ concentration contributed to the cellular defect of TNNI3 p.R193H mutation. This Ca${}^{2+}$-independent mechanical force was blocked by chronic inhibition of the interaction between actin and myosin proteins [92]. Another heterozygous p.R204H mutation in exon 8 of TNNI3 was identified in a young female patient with pure RCM who had undergone heart transplantation at the age of 23 [20]. The specific mechanism of how p.R204H mutation induces primary RCM is still unclear, although the phenotype and clinical condition deteriorated rapidly. Additionally, two novel disease-causing p.L144H and p.R170Q missense mutations, both present in exon 7 of TNNI3, were found in a family with four affected patients and a single unrelated patient essentially associated with RCM [21]. The p.L144H mutation was located in the first actin-binding domain and overlapped with the ATPase inhibitory domain. The p.R170Q mutation was located in the second actin-binding domain [93]. These mutations within the actin-binding domain have been presented to cause excessive inhibition in troponin I actomyosin ATPase activity [86]. Some studies at the laboratory suggest that these mutations could weaken the ability of the troponin complex to sufficiently inhibit the cross-bridge attachment when muscle cells are at the relaxation phase, which significantly decreased the rate of muscle relaxation [94, 95]. The change costs higher energy to return to the pre-contractile basal state [21]. Furthermore, a pathogenic p.P150S in exon 7 of TNNI3 responsible for RCM was confirmed in a Chinese family [22]. This mutation is in the actin and the N-terminal of the TnC binding domain where the configuration of cTnC and cTnI returns to the status before constriction and cTnI binds to actin again as a consequence of decreased Ca${}^{2+}$ concentration. However, the process fails to complete once mutation occurs in this domain, such as p.P150S, resulting in impaired diastolic filling in patients with RCM.

Besides missense mutations, deletion mutations in TNNI3 are also responsible for the development of RCM in a tiny percentage of patients. A novel deletion mutation of two nucleotides g.4789_4790delAA in exon 7 of TNNI3 was identified in RCM individual [23]. This mutation contributed to a frameshift and the presence of a premature termination codon at amino acid site 209 (E177fsX209). Another index patient diagnosed with RCM at the age of 23 and died due to progression of congestive heart failure at the age of 28 indicated a deletion of one nucleotide g.4762delG in exon 7 of TNNI3. This deletion also induced a frameshift in residue 168 and the introduction of a premature termination codon at site 176 (D168fsX176) [24]. According to laboratory tests, this mutation resulted in the truncation of the C-terminal part of cTnI and an approximate 50% decrease in total cTnI, likely leading to a nearly total deficiency of the second actin TnC binding domain. The damage of the inhibitory effect of the Tn-Tm complex on thin filaments could cause impaired myocardium relaxation and restrictive filling [24].

Collectively, the integrity of the cTnI is essential for conformation of the Tn complex in myofilament and the inhibition of actomyosin ATPase activity. To dissect the pathogenic cellular mechanisms resulting from TNNI3 mutations to identify the cause of RCM is scientifically and clinically important.

TNNT2 gene encodes the Tm-binding subunit of the Tn complex in the heart, which acts as a regulator of striated muscle contraction in response to differential intracellular Ca${}^{2+}$ concentration [96]. It is well established that the association between pathogenic TNNT2 mutations and risk of cardiomyopathies [25].

The first case of RCM caused by a de novo mutation of TNNT2 was reported in a 12-month-old girl [97]. This infantile case had experienced recurrent episodes of sinus bradycardia and tachycardia, malignant ventricular arrhythmias and hemodynamic instability. She received extracorporeal membrane oxygenation therapy, followed by a biventricular assist device insertion and subsequently underwent heart transplantation [97]. Genetic testing revealed a novel deletion mutation c.285_287GGA in exon 9 of TNNT2, resulting in deletion of glutamine in 96 amino acid residual (p.96delE). The p.96delE mutation is located in the highly conserved domain. It induces the deficiency of a negative charge in the coiled-coil region, affecting the TnT-Tm-actin complex’s interactions [98]. Following experimental results demonstrated that p.96delE mutation significantly increased the Ca${}^{2+}$ sensitivity in fibres reconstituted with the adult and fetal TnT isoforms. However, the effect was enhanced in adult Tn protein [99]. Another heterozygous in-frame double deletion mutation (c.297-302AATGAG) in exon 9 of TNNT2 was reported in an RCM pediatric patient. That led to the deletion of asparagine and glutamic acid, two highly conserved amino acids, at positions 100 and 101, respectively (p.100-101delNE) [26]. This case’s clinical condition deteriorated rapidly with frequent chest pain and dyspnea, and the patient ultimately received a heart transplant 15 months after initial presentations. Histology indicated mild muscle hypertrophy, interstitial fibrosis and disarray of the myocytes. It must be mentioned that those observations revealed a certain overlap of restrictive and hypertrophic phenotypes that coexisted in this RCM case.

Some missense mutations proved to be associated with RCM. In a large family with autosomal dominant cardiomyopathy, the c.236T$>$A missense mutation in exon 8 of TNNT2 led to the substitution of isoleucine (I) with asparagine (N) at amino acid position 79 (p.I79N) [13]. RCM caused by this mutation often complicated massive biatrial enlargement, markedly abnormal diastolic function, subsequent sinus bradycardia and progression to complete heart block, and even needed radiofrequency ablation and pacemaker/cardioverter-defibrillator implantation therapy in some patients. A transgenic mice model with targeted human cTnT (TNNT2 p.I79N) protein expression showed enhanced calcium-activated force generation and ATPase activity without muscle hypertrophy. The rate of Ca${}^{2+}$ dissociation from TnC during diastole decreases, and the baseline muscle tension increases, resulting in slower relaxation, the elevation of end-diastolic pressure and subsequent diastolic heart failure [100, 101]. Another index RCM case induced by a novel nucleotide substitution g.9718G$>$A in exon 10 of TNNT2 was associated with myocyte vacuolation according to the histology of proband’s explanted heart. This disorder is commonly observed in TnT-mutation related cardiomyopathy [23, 27]. The underlying pathogenicity of this new variant remains to be elucidated.

Therefore, the identified TNNT2 mutations, such as p.100-101delNE and p.I79N, associated with RCM often occur in a TnT binding fragment corresponding to residues 70–170 in the N-terminal domain. This finding suggests the existence of a mutational hotspot region in TNNT2 where mutations may result in impaired Tm-dependent functions of cTnT [102].

Cardiac troponin C (cTnC) consists of two globular EF-hand (the most common calcium-binding motif) domains and a flexible linker. The calcium-sensing part of the Tn complex is troponin C encoded by TNNC1 in both cardiac muscle and slow skeletal muscle. There are two high-affinity calcium-binding sites in the C-domain of cTnC where are often occupied by Ca${}^{2+}$ in physiologic conditions [103].

Previously, mutations in TNNC1 have been associated with HCM or DCM. Nowadays, the evidence indicated that a compound heterozygous mutation p.A8V (c.C23T) and p.D145E (c.C435A) in TNNC1 inducing fatal RCM was described in a pediatric proband who inherited the mutation from her unaffected paternal grandmother and maternal grandfather, respectively [28]. The younger sister of this proband, who carried the same genetic background, initially showed congenital HCM, evolved to RCM, subsequently occurred with heart failure and death. This phenomenon suggests that RCM induced by the compound heterozygosity p.A8V and p.D145E is combined with a young-onset marked restrictive physiology, familial history of sudden cardiac death and gradually evolves into septal hypertrophy. The p.A8V mutation alone caused a more open cTnC N-domain conformation, presumably increasing interactions with the switch region of cTnI [104], while the p.D145E mutation altered Ca${}^{2+}$ bind by the C-domain of cTnC [105]. It appeared not compatible with the fact that the grandparents of the proband who carried single p.A8V or p.D145E allele were unaffected. The seemingly contrasting finding might be explained by the possibility that the single mutation was haploinsufficiency to cause a complete penetrance. However, the combination of compound heterozygotes p.A8V and p.D145E resulted in a more severe phenotype of RCM. Experimental results demonstrated that the major abnormality induced by p.A8V and p.D145E mutations at the same time was the decreased Ca${}^{2+}$ off-rate, altered muscle relaxation and impairing diastolic function [106, 107].

As described above, myosin is a hexameric contractile protein containing two heavy chains (MHC, encoded by MYH7 in the heart) associated with four light chains (MLC). The four MLCs are classified as two regulatory light chains (encoded by MYL2 in the heart) and two essential light chains (encoded by MYL3 in the heart). The C-terminal part of each MHC is $\alpha$ helical, whereas its N-terminal part folds into a globular head region called subfragment 1 (S1). The S1 contains a motor domain binding to actin. It hydrolyses ATP and a neck domain composed of a regulatory and essential light chain, respectively, functioning as a lever for filament sliding in contraction [108, 109].

A de novo heterozygous mutation p.P838L was firstly identified in MYH7 in an infantile RCM case. The clinical presentation of this proband was characterized by early-onset, mild hypertrophy of the left ventricle and a very short evolution to death [14]. The p.P838L mutation is located in an extremely conserved hinge segment between the rod region and the globular head region of myosin protein. The marked restrictive physiology might result from the myosin head region’s impaired flexion during the relaxation cycle. However, in a p.P838L myosin transgenic Drosophila melanogaster model, the heart morphology and cardiac function was normal, although the p.P838L mutant myosin increased basal ATPase, actin sliding velocity, rotational flexibility and the average angle of two heads in vitro [110]. On the one hand, the seemingly different findings might result from the possibility that Drosophila myosin protein is less sensitive to the p.P838L perturbation than humans. On the other hand, the identification of the human pathogenic mutations involved sequencing of select candidate genes. Hence, it is possible that a mutation in another genetic locus, alone or in conjunction with P838L myosin, is responsible for the severe phenotype observed in the human patients [110]. Another missense mutation, p.G768R in exon 21 of MYH7, also was found in a pediatric RCM case [29]. The p.G768R locates in a highly conserved region across species and has previously been reported as a disease-causing mutation associated in adults with HCM [111]. It suggests that the phenotypic manifestations of MYH7 mutations in children, especially young ones, are different from adult ones. Further investigations are needed to determine whether other untested genetic mutations or sensitive indicators functioned as potential contributors to the severity and age of onset. A novel MYH7 p.R721K mutation was found in an RCM proband, who died at 47-year old due to progressive congestive heart failure, and her young son both showed biatrial enlargement, normal wall thickness and restrictive features. Yet, her other non-carrier son did not have these features [30]. The p.R721K mutation located in the converter domain of MYH7 affects myosin’s ATPase activity. RCM induced by MYH7 mutation in this domain is associated with severe diastolic heart failure, high rates of atrial fibrillation, stroke, poor prognosis and even sudden cardiac death [30]. Another p.Y386C mutation was reported in exon 13 of MYH7, which was previously seen in an infant with de novo HCM by the laboratory. The index case died at the age of 18 months, and the autopsy findings presented RCM, not HCM [31]. Interestingly, this is the first observation in a patient with RCM overlapped myocardial bridging under an MYH7 mutant background.

RCM caused by MLC-related mutation was firstly reported in an El-Salvadoran 22-year-old female. The patient underwent recurrent syncope and severe heart failure [32]. There were homozygous mutations of MYL3 p.E143K (c.427G$>$A), combined with a novel heterozygous mutation of MYL2 p.G57E (c.170G$>$A). Her mother, who carried a double heterozygous MYL3 p.E143K and MYL2 p.G57E, showed a normal echocardiogram and electrocardiogram examinations. According to this phenomenon, the homozygous MYL3 p.E143K was highly considered to contribute to RCM in the proband [32].

The $\alpha$-tropomyosin is encoded by TPM1 and plays a crucial role in actin regulation and stability, participating in fundamental functions in heart development. Mutations in TPM1 cause dominantly inherited cardiomyopathies [112]. Almost all recently reported TPM1 variants are missense mutations that resulted in a single amino acid substitution.

A novel homozygous missense mutation TPM1 p.N279H (c.835A$>$C) was found in an Italian RCM case. The endomyocardial biopsy showed mild myocyte hypertrophy and no evidence of amyloid or iron deposition [32]. This proband’s father carried heterozygous p.N279H mutation and was diagnosed with HCM in the absence of restrictive physiology. In 2021, the compound heterozygous TPM1 variants p.E62Q (c.184G$>$C) and p.M281T (c.842T$>$C) were identified in a child with RCM for the first time [33]. This proband was diagnosed with RCM at the age of 6, received orthotopic heart transplantation at 12-year old, and reached adult age without cardiovascular events. In addition, the family members of the proband carrying one of these two mutations presented HCM phenotypes. Following tests suggested that TPM1 mutations resulted in time-dependent and progressive deterioration of cardiomyocyte CaT amplitudes. Yet, the reduced CaT amplitudes and the deficient sarcomeric structures are independent of the TPM1 mutations and the clinical phenotypes of cardiomyopathies [33].

Actin is a highly conserved protein and encoded by ACTC1. Mutations in this gene have been phenotypically associated with various cardiac abnormalities. A novel p.D313H mutation (g.4642G$>$C) in exon 5 of ACTC1 was observed in an individual with RCM [23]. Interestingly, the proband’s father died from DCM after heart transplantation and the older sister was diagnosed with overlapping phenotypes of RCM and DCM. The p.D313H was located in the immobilized region of the actin filament, acting as an important tropomyosin-binding site [113]. The specific mechanism by how the p.D313H mutation induced various clinical phenotypes of cardiomyopathy remains unclear.

The MYBPC3 gene encodes the cardiac isoform of myosin-binding protein C (MyBPC), a myosin-associated and large multi-domain protein. The role of MyBPC in the sarcomere regulation is not yet fully understood. Previously, MYBPC3 mutations were demonstrated highly related to familial HCM [114]. However, a nonsense mutation MYBPC3 p.Q463X (c.1387C$>$T) was identified in a multigenerational family with three adult RCM patients. Moreover, another missense mutation MYBPC3 p.E334K (c.1000G$>$A) was observed in an unrelated patient [34]. A zebrafish model with genetic knockdown of MYBPC3 showed ventricular hypertrophy and diastolic heart failure manifestations, including decreased diastolic relaxation velocity, pericardial effusion and dilatation of the atrium [115]. It is noted that primary RCM caused by MYBPC3 mutation is associated with severe diastolic dysfunction, yet the long-term prognosis is still obscure [34].

Desmin is encoded by DES and functions as the chief intermediate filament of the skeletal and cardiac tissue connecting the Z-bands to the subsarcolemmal cytoskeleton. Cardiomyopathies caused by DES mutations often present severe restrictive physiology, syncope, sudden cardiac death due to conduction defect and overlap with a heterogeneous group of skeletal myopathies [116].

In four unrelated probands with RCM complicated with the atrioventricular block (AVB), there were three novel mutations p. R16C, p.T453I, a 10-bp deletion at the exon-intron boundary of exon 3 and one known heterozygous mutation p.R406W identified in DES [35]. The novel p.R16C mutation was associated with a recessive phenotype due to the absence of RCM in three heterozygous carriers. The novel p.T453I mutation is located in the highly conserved 9-amino acid motif among type III intermediate filaments acting as desmin interaction with other cytoskeletal proteins [117]. The new 10-bp deletion at the exon-intron boundary of exon 3 damaged the exon 3 donor splice site, predicting the loss of 32 amino acids and the accumulation of desmin-positive material [118]. The known p.R406W mutation was located in the C-terminal of the desmin core domain and associated with early-onset severe cardiac and skeletal myopathy [119]. Although the probands carry different mutations affecting different domains, all shared the identical cardiac phenotypes of RCM in combination with AVB [35]. Another novel homozygous missense mutation DES p.Y122H (c.364T$>$C) was also reported in the index patient with RCM plus AVB [36]. Following experimental results in vitro revealed a severe filament assembly defect of mutant DES protein. The novel DES p.E413K mutation was identified in a family with pure RCM, including three affected and five at-risk members. The pathogenicity of p.E413K mutation at a highly conserved end of the alpha-helical rod domain might induce potential disruption of intramolecular interactions and inability of filamentous cellular network [37]. Recently, in an index patient with RCM in combination with atrial fibrillation, there was a heterozygous mutation c.735G$>$C in DES. This mutation affected the last base pair of exon 3 and caused a splice site defect. RCM caused by this mutation showed right heart failure, massive dilation of the right atrium and recurrent atrial fibrillation [38].

The MYPN gene encodes myopalladin protein connecting structural regulatory molecules by translocation from the Z-lines and I-bands to the cardiomyocyte nucleus [120]. Mutations in MYPN associated with DCM, HCM and RCM have been reported. MYPN p.Q529X mutation was identified in siblings with RCM, yet their carriers’ mother was not affected. Different phenotypes were observed in family members carrying the same mutation [19]. The phenomenon of reduced penetrance of p.Q529X mutation might be explained by the possibility that the patients with RCM had other disease-associated mutations inherited from their father and absent from their mother. The knock-in heterozygote MYPN p.Q526X mutant mice revealed the diastolic dysfunction and restrictive physiology. There was preserved systolic function without overt hypertrophic remodeling. The phenotype of mutant mice resembles RCM induced by p.Q529X in humans [121].

TTN (also called titin) gene encodes the largest human protein consisting of 364 exons and approximately 38,000 amino acid residues with a molecular weight of 4200 kDa. The TTN protein provides architectural support and sarcomeric organization during muscle contraction [122]. Mutations in TTN refer to the different phenotypes of cardiomyopathies and missense variants are very common and frequently benign in DCM [123]. A linkage analysis study identified a missense mutation TTN p.Y7621C (c.22862A$>$G) in a family with RCM involving six affected individuals aged 12–35 years [11]. The p.Y7621C mutation is located in titin’s most highly conserved A/I junction region, connecting the compliant I-band and the rigid thick filament bound A-band. The clinical presentations of RCM induced by TTN mutation showed severe diastolic dysfunction overlapped with atrial fibrillation and thromboembolic phenomena.

Filamin C is an actin-cross-linking protein encoded by FLNC in heart muscle. Pathogenic mutations in FLNC have been reported to cause dominant isolated cardiomyopathy phenotype. The prevalence of patients carrying a unique FLNC pathogenic mutation in a cohort was evaluated 8% in RCM [124]. It has been identified that two novel missense mutations, FLNC p.S1624L and p.I2160F, were associated with familial RCM. It was suspected that FLNC and DES mutations shared similar pathological mechanisms due to identical behaviour of cytoplasmic aggregation [39].

Mutation in BAG3 is a rare cause of RCM. Recently, a heterozygous mutation p.P209L (c.626C$>$T) in exon 3 of BAG3 was found in a 15-year-old girl. The proband showed severe myopathy, neuropathy, asymptomatic long QT syndrome and late-onset RCM [40]. The BAG3 p.P209L mutation was also present in another index patient who suffered from severe myofibrillar myopathy and RCM [41].