Analysis of Contractile Function of Permeabilized Human Hypertrophic Cardiomyopathy Multicellular Heart Tissue

Abstract

Background : Many forms of hypertrophic cardiomyopathy (HCM) show an increased myofilament Ca 2+ sensitivity. This observation has been mainly made in HCM mouse models, myofilament systems, and cardiomyocytes. Studies of multicellular tissues from patients with different HCM-associated gene mutations are scarce. We investigated Ca 2+ sensitivity in multicellular cardiac muscle strips of HCM patients. We furthermore evaluated the use of epigallocatechin-3-gallate (EGCg), a Ca 2+ desensitizer. Methods : After strip isolation from cardiac tissues with single ( MYBPC3 , MYH7) or double heterozygous mutations ( MYBPC3/FLNC, MYH7/LAMP2, MYBPC3/MYH7 ) and permeabilization, we performed contractility measurements ±EGCg. We furthermore evaluated gene expression with a customized heart failure gene panel using the NanoString technology. Results : F max tended to be higher in HCM than in non-failing (NF) control strips and in single than in double heterozygous strips. Ca 2+ sensitivity was higher by trend in most HCM vs. NF strips and by trend in tissues with double vs. single heterozygous mutations. EGCg desensitized myofilaments to Ca 2+ in most of the strips and tended to induce a more pronounced shift in strips with truncating than missense or single than double heterozygous mutations. Gene expression analysis revealed lower ATP2A2 , PPP1R1A, and FHL2 and higher NPPA , NPPB, COL1A1, CTGF, and POSTN marker levels in HCM than in NF tissues . NPPA , NPPB , ACTA1 , CTGF , COL1A1 , and POSTN levels were higher in tissues with missense than truncating mutations. Conclusion : We report an increased myofilament Ca 2+ sensitivity in native multicellular cardiac HCM strips, which by trend was more pronounced in samples with double heterozygous mutations. EGCg could have differential effects depending on the underlying genetic status (single vs. double heterozygous) and type (missense vs. truncating).

Translational investigation of electrophysiology in hypertrophic cardiomyopathy

“…6 D). This suggests that this MYBPC3 mutation may prolong APD and therefore contribute to arrhythmias in the HCM patient, which could be related to the previously measured higher myofilament Ca 2+ sensitivity [ 47 ]. For the other HCM samples, however, the APD did not significantly differ to samples from patients with aortic stenosis.…”

Multi-omics integration identifies key upstream regulators of pathomechanisms in hypertrophic cardiomyopathy due to truncating MYBPC3 mutations

“…We further selected an example of one down-regulated and one up-regulated gene and examined them in more detail to demonstrate the strength of our integrative omic analysis. ATP2A2 , one protein-coding gene from the overlapping candidates, encodes the sarcoplasmic reticulum Ca2+ -ATPase pump SERCA2a and plays a critical role in the regulation of calcium handling [ 14 ]. We identified ATP2A2 as one of the major candidates in discriminating HCM hearts from controls in our integrated H3K27ac ChIP-seq and RNA-seq analysis (Fig.…”

“…Among these, ATP2A2 /SERCA2a, the cardiomyocyte-specific marker, transports cytosolic calcium into the sarcoplasmic reticulum and subsequently regulates the contraction and relaxation of cardiomyocytes [ 18 ]. Suppressed ATP2A2 /SERCA2a expression is associated with impaired relaxation in cardiomyocytes and contributes to diastolic dysfunction in HCM [ 14 , 19 ]. Consistent with previous findings, we also showed lower mRNA and protein levels in HCM hearts compared to controls.…”

Phosphomimetic cardiac myosin-binding protein C partially rescues a cardiomyopathy phenotype in murine engineered heart tissue

“…The prevention of the EHT disease phenotype after Mybpc3 D282 and S282 gene transfer was expected in both conditions since the amount of cMyBP-C protein was completely restored, and we recently showed that ≥73% of wild-type cMyBP-C in a Mybpc3 null background is enough to overcome the disease phenotype 17 . Increased myofilament [Ca 2+ ] sensitivity is a well-accepted hallmark of HCM as seen in human samples 11,36,47,48 and in HCM animal models 15,17,35,49–53 . Accordingly, KI-NT showed higher sensitivity to external [Ca 2+ ] than WT EHTs.…”

“…Force-calcium relationship data were analyzed using the Hill equation, with EC 50 as the free Ca 2+ concentration, which yields 50% of the maximal force and nH representing the Hill coefficient. Curves were fitted to the data points and force-pCa relationship comparison was done by using extra sum-of-squares F-test 48 . A value of P < 0.05 was considered statistically significant.…”