Biology subjects

Graham, R. M.

Publications and source records attributed to Graham, R. M..

4 recordsLinked to original sources

bioRxiv2021

A standardized method to purify cardiomyocytes from individual mouse hearts of any age

Primary cardiomyocytes are invaluable for understanding postnatal heart development. However, a universal method to obtain freshly purified cardiomyocytes without using different agedependent isolation procedures and cell culture, is lacking. Here, we report the development of a standardised method that allows rapid isolation and purification of high-quality cardiomyocytes from individual neonatal through to adult C57BL/6J murine hearts. Langendorff retrograde perfusion, which is currently limited to adult hearts, was adapted for use in neonatal and infant hearts by developing an easier in situ aortic cannulation technique. Tissue digestion conditions were optimised to achieve efficient digestion of hearts of all ages in a comparable timeframe (<14 min). This resulted in a high yield (1.56-2.2x106 cells/heart) and viability (~70-100%) of cardiomyocytes post-isolation. An immunomagnetic cell separation step was then applied to yield highly purified cardiomyocytes (~95%) as confirmed by immunocytochemistry, flow cytometry, and qRT-PCR. For cell-type specific studies, cardiomyocyte DNA, RNA, and protein could be extracted in sufficient yields to conduct molecular experiments. We generated transcriptomic datasets for neonatal cardiomyocytes from individual hearts, for the first time, which revealed nine sex-specific genes (FDR<0.05) encoded on the sex chromosomes. Finally, we also developed an in situ fixation protocol that preserved the native cytoarchitecture of cardiomyocytes (~94% rodshaped post-isolation), and used it to evaluate cell morphology during cardiomyocyte maturation, as well as capture spindle-shaped neonatal cells undergoing cytokinesis. Together, these procedures allow molecular and morphological profiling of high-quality cardiomyocytes from individual hearts of any postnatal age.

cell biology↗

bioRxiv2020

The Ca2+-activated cation channel TRPM4 is a positive regulator of pressure overload-induced cardiac hypertrophy

Pathological left ventricular hypertrophy (LVH) is a consequence of pressure overload caused by systemic hypertension or aortic stenosis and is a strong predictor of cardiac failure and mortality. Understanding the molecular pathways in the development of pathological LVH may lead to more effective treatment. Here, we show that the transient receptor potential cation channel subfamily melastatin 4 (TRPM4) ion channel is an important contributor to the mechanosensory transduction of pressure overload that induces LVH. In mice with pressure overload induced by transverse aortic constriction (TAC) for two weeks, cardiomyocyte TRPM4 expression was reduced, as compared to control mice. Cardiomyocyte-specific TRPM4 inactivation reduced by ~50% the degree of TAC-induced LVH, as compared with wild type (WT). In WT mice, TAC activated the CaMKII{delta}-HDAC4-MEF2A but not the calcineurin-NFAT-GATA4 pathway. In TRPM4 knock-out mice, activation of the CaMKII{delta}-HDAC4-MEF2A pathway by TAC was significantly reduced. However, consistent with a reduction in the known inhibitory effect of CaMKII{delta} on calcineurin activity, reduction in the CaMKII{delta}-HDAC4-MEF2A pathway was associated with partial activation of the calcineurin-NFAT-GATA4 pathway. These findings indicate that the TRPM4 channel and its cognate signalling pathway are potential novel therapeutic targets for the prevention of pathological pressure overload-induced LVH. Significance statementPathological left ventricular hypertrophy (LVH) occurs in response to pressure overload and remains the single most important clinical predictor of cardiac mortality. Preventing pressure overload LVH is a major goal of therapeutic intervention. Current treatments aim to remove the stimulus for LVH by lowering elevated blood pressure or replacing a stenotic aortic valve. However, neither of these interventions completely reverses adverse cardiac remodelling. Although numerous molecular signalling steps in the induction of LVH have been identified, the initial step by which mechanical stretch associated with cardiac pressure overload is converted into a chemical signal that initiates hypertrophic signalling, remains unresolved. Here, we demonstrate that the TRPM4 channel is a component of the mechanosensory transduction pathway that ultimately leads to LVH.

physiology↗

bioRxiv2020

Cardiac Gq receptors and calcineurin activation are not required for the hypertrophic response to mechanical left ventricular pressure overload

RationaleGq-coupled receptors are thought to play a critical role in the induction of left ventricular hypertrophy (LVH) secondary to pressure overload, although mechano-sensitive channel activation by a variety of mechanisms has also been proposed, and the relative importance of calcineurin- and calmodulin kinase II (CaMKII)-dependent hypertrophic pathways remains controversial. ObjectiveTo determine the mechanisms regulating the induction of LVH in response to mechanical pressure overload. Methods and ResultsTransgenic mice with cardiac-targeted inhibition of Gq-coupled receptors (GqI mice) and their non-transgenic littermates (NTL) were subjected to neurohumoral stimulation (continuous, subcutaneous angiotensin II (AngII) infusion for 14 days) or mechanical pressure overload (transverse aortic arch constriction (TAC) for 21 days) to induce LVH. Candidate signalling pathway activation was examined. As expected, LVH observed in NTL mice with AngII infusion was attenuated in heterozygous (GqI+/-) mice and absent in homozygous (GqI-/-) mice. In contrast, LVH due to TAC was unaltered by either heterozygous or homozygous Gq inhibition. Gene expression of atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and -skeletal actin (-SA) was increased 48 hours after AngII infusion or TAC in NTL mice; in GqI mice, the increases in ANP, BNP and -SA in response to AngII were completely absent, as expected, but all three increased after TAC. Increased nuclear translocation of nuclear factor of activated T-cells c4 (NFATc4), indicating calcineurin pathway activation, occurred in NTL mice with AngII infusion but not TAC, and was prevented in GqI mice infused with AngII. Nuclear and cytoplasmic CaMKII{delta} levels increased in both NTL and GqI mice after TAC but not AngII infusion, with increased cytoplasmic phospho- and total histone deacetylase 4 (HDAC4) and increased nuclear myocyte enhancer factor 2 (MEF2) levels. ConclusionCardiac Gq receptors and calcineurin activation are required for neurohumorally mediated LVH but are not required for LVH induced by mechanical pressure overload (TAC); the latter is mediated by activation of the CaMKII-HDAC4-MEF2 pathway.

molecular biology↗

bioRxiv2020

The N-terminus of GPR37L1 is proteolytically processed by matrix metalloproteases

GPR37L1 is an orphan G protein-coupled receptor expressed exclusively in the brain and linked to seizures, neuroprotection and cardiovascular disease. Based upon the observation that fragments of the GPR37L1 N-terminus are found in human cerebrospinal fluid, we hypothesized that GPR37L1 was subject to post-translational modification. Heterologous expression of GPR37L1-eYFP in either HEK293 or U87 glioblastoma cells yielded two cell surface species of approximately equivalent abundance, the larger of which is N-glycosylated at Asn105. The smaller species is produced by matrix metalloprotease/ADAM-mediated proteolysis (shown by the use of pharmacological inhibitors) and has a molecular weight identical to that of a mutant lacking the entire N-terminus, Δ122 GPR37L1. Serial truncation of the N-terminus prevented GPR37L1 expression except when the entire N-terminus was removed, narrowing the predicted site of N-terminal proteolysis to residues 105-122. Using yeast expressing different G protein chimeras, we found that wild type GPR37L1, but not Δ122 GPR37L1, coupled constitutively to Gpa1/Gαs and Gpa1/Gα16 chimeras, in contrast to previous studies. We tested the peptides identified in cerebrospinal fluid as well as their putative newly-generated N-terminal ‘tethered’ counterparts in both wild type and Δ122 GPR37L1 Gpa1/Gαs strains but saw no effect, suggesting that GPR37L1 does not signal in a manner akin to the protease-activated receptor family. We also saw no evidence of receptor activation or regulation by the reported GPR37L1 ligand, prosaptide/TX14A. Finally, the proteolytically processed species predominated both in vivo and ex vivo in organotypic cerebellar slice preparations, suggesting that GPR37L1 is rapidly processed to a signaling-inactive form. Our data indicate that the function of GPR37L1 in vivo is tightly regulated by metalloprotease-dependent N-terminal cleavage.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗