The mechanisms responsible for the onset of iDCM and its evolution to overt HF and death are currently unknown. The documentation in the present study that fibrillar deposits with the formation of tangle- and plaque-like structures within the myocardium occur in the early and late stages of the disease point to protein misfolding as an important determinant of the pathogenesis of iDCM. Protein misfolding impairs cell function in neurodegenerative disorders and other chronic diseases suggesting that a similar effect may be operative in the human heart18,26,27
. Although the negative impact of protein aggregates on the mechanical and Ca2+
handling properties of human myocytes was not tested, these deposits modified the functional behavior of normal adult mouse myocytes supporting the notion that similar consequences may occur on human myocytes.
Protein degradation characteristically occurs with myocardial aging. Senile systemic amyloidosis occurs in 25% of individuals 80 years of age and older28
. Amyloid-like deposits are described in non-diseased individuals between the sixth and ninth decade of life29,30
. In this report, aggregates were also markedly pronounced in relatively younger patients at early stages of HF, excluding age and disease stage as accounting for protein misfolding in our iDCM population. On the other hand, the presence of protein aggregates, common in advanced age, may suggest that iDCM can be an early senility of the heart, similarly to AD where the common senile dementia occurs at early age.
We then evaluated whether the aggregates simply represent a tombstone of discarded material or are they relate to the disease. Protein aggregates are composed of misfolded proteins at various maturation stages, from monomeric peptides to intermediate oligomers and complex fibrillar structures. Intermediate oligomers have been shown to be present in the brain of patients with AD and, in neurons, to be associated with cell toxicity31,32
. Here we showed that intermediate oligomers are also present in the myocardium of patients with iDCM with a distribution similar to what is observed in the brain of patients with AD17
. We also demonstrated for the first time that, on cardiomyocytes, oligomers promote an increase in systolic [Ca2+
]that may lead to cell dysfunction and death33–35
. An increase in cytosolic Ca2+
in the failing heart is coupled with Ca2+
depletion in the sarcoplasmic reticulum which, in turn, favors protein misfolding26,36,37
. In the failing heart, Ca2+
dishomeostasis and contractile dysfunction are well known abnormalities and are characterized by changes in excitation-contraction coupling proteins, with SERCA2a playing a key role37
Several proteins involved in the control of Ca2+
cycling have been shown to be substrates of presenilin/γ secretase11
. PS has been recently shown to interact with SERCA2b attenuating its function when PS is downregulated24
. Importantly, PS1 also co-immunoprecitpitate with SERCA2a in our iDCM cases suggesting that PS1 may also depress SERCA2a thereby contributing to the observed alterations in Ca2+
homeostasis and myocyte performance in HF. Sequencing of PSEN1
led to the identification of 26 DNA variants including two novel PSEN1
gene regulatory region variants, -92delC and -21G>A, in our iDCM patient sample. These two previously unknown variants significantly reduced mRNA and protein expression of PS1 indicating loss of function of the genetic variants. Thus these variants likely influence Ca2+
homeostasis by altering SERCA2a function.
In our cases of sporadic iDCM, two PSEN
missense mutations (PSEN
1 D333G and PSEN2
R62H) previously described in familial DCM9
were detected in addition to the variants in the PSEN
promoter. A PSEN1
missense polymorphism E318G was also found. This mutation causes amino acid changes from polar acidic Glutamic acid or Aspartic acid to non-polar neutral Glycine, similar to the PSEN1 D333G mutation. E318G and D333G are located 15 amino acids apart within a “hot spot” in a large hydrophilic cytoplasmic loop between transmembrane domains six and seven. This region contains critical functional domains which are processed by endoproteolysis38
and are essential for the interaction between PS1 and other proteins39
. The PSEN2
R62H mutation identified in patients 1 and 17 is located in a “hot spot” of the NH2 terminus and has been associated with AD22
and breast cancer40
R62H enhances PS2 degradation, reduces PS2 stability and compromises Notch function40
The two presenilin missense substitutions, E318G and R62H observed in iDCM are present in highly divergent regions of PS1 and PS2; PS1 and PS2 proteins share extensive sequence identity along their entire length with the exception of the NH2-terminal domain and the second half of the hydrophilic loop region. Thus, the E318G and R62H missense substitutions may play an important role in the pathogenesis of a subset of patients with iDCM by modulating different functions of the presenilin proteins. An important aspect is whether PSEN variants could affect APP processing in the heart and determine the accumulation of amyloid aggregates. Since the sequence variants we observed are loss of function any effect on APP processing would reduce the Aβ production. Aβ level was tested by immunohistochemistry in our samples and no difference in Aβ expression was present in iDCM compared to controls.
Four of the seven Caucasian female with iDCM possessed either a PSEN1
promoter region variant or a PSEN2
missense mutation. This gender dependent cardiac genotype is consistent with the higher prevalence of AD in women than men41
. Estrogen deficiency typical in postmenopausal women may be implicated since cardiomyocytes and fibroblasts contain estrogen receptors and estrogens regulate the expression of specific cardiac genes42
In conclusion, we have provided the first evidence that protein misfolding is implicated in the etiology and pathogenesis of iDCM in humans. The formation of oligomeric fragments has toxic consequences on cardiomyocytes, which are mediated by alterations in Ca2+ homeostasis. Additionally, missense mutations in the PSEN1 and PSEN2 genes alter presenilin expression and may alter its interaction with proteins involved in excitation-contraction coupling. Further studies in a larger cohort and in animal models will be needed to evaluate the role and mechanisms of these sequence variants and the respective protein expression in the familial and sporadic cases compared with the non-failing population. Furthermore cardiac function is needed to be assessed in AD patients and the AD unaffected family members.
The elucidation of these new pathogenetic mechanisms will facilitate the advancement of novel strategies for early diagnosis and treatment of iDCM.
Heart failure (HF) is a progressive and ultimately fatal disease, which represents a leading public health problem worldwide. Despite substantial advances in the clinical management of HF, the only current permanent therapeutic option is heart transplantation. However, the limited supply of functional organs for transplantation and the age threshold for the surgical intervention constrains the scope of this treatment modality. The majority of cases of HF are ischemic in origin. The second most frequent origin of non-ischemic HF is “idiopathic” dilated cardiomyopathy (iDCM) where no other causative events can be recognized. Recent studies highlight the recognition of the public health importance of a large number of diseases associated with defects in the ability of proteins to fold, leading to the accumulation of cytotoxic protein deposits. These group of diseases include amyloidosis and various neurodegenerative disorders such as Alzheimer’s Disease. Preliminary evidence suggests that iDCM may be included among these misfolding diseases. Also, mutations in the same genes causing Alzheimer’s Disease in a significant percentage of cases can also be at the origin of iDCM. Interestingly, a dual mechanism seems to mediate the effect of these genetic variations: changes in the control of calcium (Ca2+) handling proteins causing defect in the contractile function, and cell damages associated with the protein aggregation process. The incidence, prognosis, and therapeutic option for iDCM may, therefore, be greatly advanced by establishing a fundamental understanding of key factors leading to the disease.