Nathalie Ringström

The cardiac extracellular matrix (ECM) is involved in several pathological conditions, and age itself is also associated with certain pathological changes in the heart: it gets larger and stiffer, and it develops an increased risk of abnormal intrinsic rhythm. Many of these changes are directly related to the ECM, yet the proteomic composition of the ECM and how it changes with age is not fully resolved. As part of this PhD project, mass spectrometry (MS) studies were deployed to decipher the proteomic composition of the cardiac ECM of young and old mice. Differential expression analysis revealed that 237 proteins were found to be significantly differentially expressed with age. A number of proteins (MMP9, S100a9, vwa3a, ctsd, ccl8) were more than threefold increased in aged mice and the overall collagen content was markedly decreased. STRING network mapping of physical associations predicted that both PLOD3 and PDGFA interact with the downregulated collagens. The results suggests that the mechanism behind age-associated atrial stiffness is not due to an increase in collagen content as previously believed, but rather an increase in cross-linking, potentially facilitated by PLOD3. Additionally, several of the differentially expressed proteins, such as COL7A1 have not previously been associated with cardiac ageing, and thus are potential drug targets for age-associated cardiac fibrosis and other age-associated cardiac conditions.

One of the significantly upregulated proteins was Sonic Hedgehog (SHH). Previous studies have suggested that SHH is cardioprotective after MI, and results from proteomic studies suggests that the protein may have protective effects in aged atria also. To evaluate how SHH affects calcium handling properties of induced pluripotent stem cell (iPSC) derived atrial cardiomyocytes, cells were treated with SHH for 48 h, and their calcium handling properties and gene expression were evaluated. SHH treatment led to a significant reduction in calcium transient frequencies and resulted in significant suppression of a number of pathways including the calcium handling, focal adhesion, cytoskeleton in muscle cells and ECM-receptor interaction pathways. As the cardiac ECM is important for the electrophysiological properties of the atria, this suggests that the reduction in calcium transient frequency is caused by altered expression of genes in the mentioned pathways. A reduced beating frequency is potentially a cardioprotective effect and the results therefore indicate that SHH may be a promising therapeutic agent to treat age associated decline in cardiac functioning. However, as SHH is capable of interacting with receptors of many different cell types, non-invasive in treatment with SHH in the form of e.g. an intravenous injection would most likely have off-target effects with potentially detrimental consequences. However, local treatment with SHH in the form of e.g. a cardiac patch would likely carry less risk. Finally, other studies have suggested that SHH may also have detrimental effects on cardiomyocytes through swift non-canonical SHH signalling, and care must therefore be taken when considering SHH as a therapeutic agent.

In conclusion, the studies described in this thesis have successfully aided deciphering of the cardiac ECM and how it changes with age, facilitating a better understanding of the complex pathophysiology of cardiac ageing. Additionally, it has provided an example of how the plethora of data yielded from the MS experiments can be used, for example, some of the identified proteins may be promising new therapeutic agents. In this manner, the in vitro studies described here further confirmed that SHH may be a suitable therapeutic agent for age associated decline in cardiac functioning, although more studies are still required.

The cardiac atrial extracellular matrix (ECM) is central to age-associated cardiac remodeling and subsequent decline in cardiac functioning. Despite this, the composition of the atrial ECM and how it changes with age is not yet known. This study utilized mass spectrometry to evaluate the composition of murine atria in young (12 weeks) and old (77 weeks) C57BL/6J mice. The tissue was decellularized, ECM and ECM-associated proteins were extracted with GuHCl, and proteins were deglycosylated to enable identification of glycosylated peptides. Two hundred and thirty-seven ECM and ECM-associated proteins were found to be significantly differentially expressed with age. Some proteins (MMP9, S100A9, VWA3A, CTSD, CCL8) were more than threefold increased with age, proteoglycans were modestly decreased, while the overall collagen content was markedly decreased. STRING network mapping of physical associations predicted that both PLOD3 and PDGFA interact with the collagens that decreased with age. The results suggest that the mechanism behind age-associated atrial stiffness is not due to an increase in collagen content as previously believed, but an increase in cross-linking, potentially facilitated by PLOD3. Additionally, several of the significant proteins have not previously been associated with cardiac aging and thus are potential drug targets for age-associated cardiac fibrosis and other age-associated conditions.

The cardiac extracellular matrix (ECM) is involved in several pathological conditions, and age itself is also associated with certain changes in the heart: it gets larger and stiffer, and it develops an increased risk of abnormal intrinsic rhythm. This, therefore, makes conditions such as atrial arrythmia more common. Many of these changes are directly related to the ECM, yet the proteomic composition of the ECM and how it changes with age is not fully resolved. The limited research progress in this field is mainly due to the intrinsic challenges in unravelling tightly bound cardiac proteomic components and also the time-consuming and costly dependency on animal models. This review aims to give an overview of the composition of the cardiac ECM, how different components aid the function of the healthy heart, how the ECM is remodelled and how it is affected by ageing.