Abstract
Key points Mutations in genes encoding cardiac troponin I (TNNI3) and cardiac troponin T (TNNT2) caused altered troponin protein stoichiometry in patients with dilated cardiomyopathy. TNNI3p.98trunc resulted in haploinsufficiency, increased Ca2+‐sensitivity and reduced length‐dependent activation. TNNT2p.K217del caused increased passive tension.A mutation in the gene encoding Lamin A/C (LMNA p.R331Q) led to reduced maximal force development through secondary disease remodelling in patients suffering from dilated cardiomyopathy.Our study shows that different gene mutations induce dilated cardiomyopathy via diverse cellular pathways. Dilated cardiomyopathy (DCM) can be caused by mutations in sarcomeric and non‐sarcomeric genes. In this study we defined the pathogenic effects of three DCM‐causing mutations: the sarcomeric mutations in genes encoding cardiac troponin I (TNNI3p.98truncation) and cardiac troponin T (TNNT2p.K217deletion; also known as the p.K210del) and the non‐sarcomeric gene mutation encoding lamin A/C (LMNAp.R331Q). We assessed sarcomeric protein expression and phosphorylation and contractile behaviour in single membrane‐permeabilized cardiomyocytes in human left ventricular heart tissue. Exchange with recombinant troponin complex was used to establish the direct pathogenic effects of the mutations in TNNI3 and TNNT2. The TNNI3p.98trunc and TNNT2p.K217del mutation showed reduced expression of troponin I to 39% and 51%, troponin T to 64% and 53%, and troponin C to 73% and 97% of controls, respectively, and altered stoichiometry between the three cardiac troponin subunits. The TNNI3p.98trunc showed pure haploinsufficiency, increased Ca2+‐sensitivity and impaired length‐dependent activation. The TNNT2p.K217del mutation showed a significant increase in passive tension that was not due to changes in titin isoform composition or phosphorylation. Exchange with wild‐type troponin complex corrected troponin protein levels to 83% of controls in the TNNI3p.98trunc sample. Moreover, upon exchange all functional deficits in the TNNI3p.98trunc and TNNT2p.K217del samples were normalized to control values confirming the pathogenic effects of the troponin mutations. The LMNAp.R331Q mutation resulted in reduced maximal force development due to disease remodelling. Our study shows that different gene mutations induce DCM via diverse cellular pathways.
Highlights
Dilated cardiomyopathy (DCM) is a cardiac disease characterized by dilatation of the left ventricle (LV) and a reduced systolic function
Among the various genes implicated in DCM are genes encoding sarcomeric proteins such as cardiac troponin I (Carballo et al 2009; van Spaendonck-Zwarts et al 2013), cardiac troponin T (Hershberger et al 2009; van Spaendonck-Zwarts et al 2013; Walsh et al 2017) and titin (Herman et al 2012; Walsh et al 2017), and genes encoding for non-sarcomeric proteins such as lamin A/C, a protein involved in nuclear stability (Parks et al 2008; van Spaendonck-Zwarts et al 2013; Walsh et al 2017)
No difference in maximal force (Fmax) (Fig. 2A) was observed in idiopathic DCM (IDCM), TNNI3p.98trunc and TNNT2p.K217del cardiomyocytes, while Fmax was significantly lower in cardiomyocytes with the LMNAp.R331Q mutation (17.9 kN m−2, data from Hoorntje et al 2016) compared to controls (28.2 kN m−2)
Summary
Dilated cardiomyopathy (DCM) is a cardiac disease characterized by dilatation of the left ventricle (LV) and a reduced systolic function. The Exome Aggregation Consortium (ExAC) recently reported that many rare variants in various sarcomeric and non-sarcomeric genes, which were assumed to be disease-causing, only have limited pathogenic burden as no or limited excess variation was found in a DCM population compared with ß60,000 reference samples (Walsh et al 2017). In this study we defined the pathological effects on cardiomyocyte function of three different DCM-causing mutations in genes encoding sarcomeric (TNNI3, TNNT2) and non-sarcomeric (LMNA) proteins
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