Functional phenotyping combined with single-cell sequencing unmasks potential genes for hypercontractility in murine HFpEF

Abstract Background We showed in a murine model of heart failure with preserved ejection fraction (HFpEF) on a cellular level a hypercontractile phenotype with an impaired functional response to adrenergic stress (Semmler L et al. Acta Physiol 2024). The potential relationship between observed phenotype and altered gene expression levels are unclear. In this work, we measured isolated adult murine ventricular myocytes (AMVM) function followed by simultaneous single cell sequencing. Methods To induce HFpEF, 12-weeks-old male C57BL6/N mice were fed with a high-fat diet (D12492, Research Diet, chow diet for control mice) and L-NAME (0.5g/l, via drinking water) for 19 weeks. Finally AMVM were isolated to examine sarcomere shortening and cellular calcium handling (Fura-2, 1Hz). In this way phenotyped individual AMVMs were picked with a micropipette setup and separately prepared for single cell sequencing (n = 15 for control and n = 16 for HFpEF). We used the well described smart-seq2 approach. We used STAR for alignment of sequencing reads to a reference genome. We focused on genes related to excitation contraction coupling and mechanical structures. Seurat was used for data analysis, a level of p0.05 was set as significant. Results HFpEF cells showed in-vitro at 1Hz pacing frequency a higher contractility with a faster relaxation time in comparison tocontrol cells. Using our approach, MYH7 (myosin heavy chain 7) and ACTN4 (actin alpha 4) were part of the overall ten most highly regulated genes. Correlating sarcomere shortening relaxation time with ACTN4 gene expression level, we found a significant positive relationship in HFpEF cells, whereas no significant relationship was observed in control cells. In HFpEF cells but not in control cells, MYH7 showed a strong trend to negatively correlate with cardiomyocyte relaxation time (p=0.06), Figure A. Regarding shortening amplitude, the gene expression levels showed a negative trend for ACTN4 but not for MYH7. Furthermore we investigated key calcium handling proteins such as RYR2, SCL8A1, ATP2A2, TNNT2. We found a significant negative correlation for RYR2 in HFpEF cells of higher expression level to lower sarcomere shortening amplitude. In contrast, there was a trend for a positive correlation in control cells. Interestingly, changes in expression levels for calcium handling proteins did not show significant changes in in-vitro calcium dynamics. Conclusion We present a combined feasible approach for in-vitro cellular function and gene sequencing in single adult cardiomyocytes. We established that faster sarcomere relaxation correlates with higher expression of MYH7 and longer sarcomere relaxation with higher ACTN4 expression levels. This novel approach links sarcomere relaxation speed to specific gene expression levels in single cardiomyocytes, providing new insights into cardiac function at the molecular level.