Circulating Factors Induce Cardiomyopathy after Burn Injury.

Cyclic guanosine monophosphate (cGMP) levels were significantly decreased in cardiomyocytes treated with 24-hour post-burn serum, and this was reversed by sildenafil treatment.

• Human cardiomyocyte cell line AC16 was divided into 4 groups: sham serum, 24 hours post-burn (24hpb) serum, sildenafil (SIL) alone, and 24hpb serum treated with SIL.
• cGMP levels were 'significantly decreased' in the 24hpb serum group compared to sham.
• Treatment with SIL 'completely reversed this change, similar to our previous in vivo work.'
• ELISA was used to measure cGMP levels across treatment groups.

Cardiac troponin I (cTnI) levels were significantly increased in cardiomyocytes treated with 24-hour post-burn serum and were reversed by sildenafil.

• cTnI levels were 'significantly increased in the 24hpb serum group.'
• Sildenafil treatment completely reversed the increase in cTnI levels.
• ELISA was used to quantify cTnI as a marker of cardiomyocyte injury.
• This finding paralleled previous in vivo work by the authors.

Cardiomyocytes treated with 24-hour post-burn serum showed significantly decreased cell viability and proliferation along with increased cytotoxicity and apoptosis.

• There was 'significantly decreased cell viability and cell proliferation' in cells treated with 24hpb serum.
• Cells showed 'increased cell cytotoxicity, cell apoptosis, and cell reactive oxygen species production' compared to sham-treated cells.
• Fluorescence microscopy was used to assess these cellular outcomes.
• Sildenafil treatment returned these responses toward sham levels.

Cardiomyocytes treated with 24-hour post-burn serum exhibited mitochondrial dysfunction characterized by decreased ATP production and compromised mitochondrial membrane integrity.

• Mitochondrial dysfunction was demonstrated by 'decreased ATP production and compromised mitochondrial membrane integrity or potential.'
• Increased mitochondrial reactive oxygen species were observed in 24hpb serum-treated cells.
• Mitochondrial function studies and fluorescence microscopy were used to assess these parameters.
• Sildenafil treatment returned mitochondrial responses 'to near sham levels.'

Seahorse and O2K respirometry confirmed mitochondrial dysfunction in post-burn serum-treated cardiomyocytes, with decreases in multiple respiratory parameters.

• Both Seahorse and O2K approaches confirmed 'mitochondrial basal respiration, proton leak, ATP production, and maximal respiration' were all decreased in 24hpb serum-treated cardiomyocytes.
• Two independent respirometry platforms (Seahorse extracellular flux analysis and O2K oxygraph) were used to validate the mitochondrial dysfunction findings.
• Sildenafil treatment reversed these respiratory deficits to near sham levels.
• Decreased proton leak was noted as part of the compromised mitochondrial membrane potential findings.

Gene expression analysis and RNA sequencing identified transcriptomic changes in cardiomyocytes exposed to post-burn circulating factors.

• Gene analysis and Illumina RNA sequencing were performed on the four treatment groups.
• These analyses were used to elucidate pathogenic mechanisms driving cardiac dysfunction after burn injury.
• GraphPad Prism 9.2.0 was used for statistical analysis of all study data.
• The study provided evidence 'for understanding the pathogenic mechanism of circulating factors released after burn injury.'

The PDE5-cGMP-protein kinase G pathway is implicated in burn-induced cardiomyocyte dysfunction, consistent with prior in vivo findings.

• Previous in vivo work had demonstrated that 'the phosphodiesterase type 5 (PDE5)-cyclic guanosine monophosphate-protein kinase G pathway is involved in burn-induced heart dysfunction.'
• The in vitro findings of decreased cGMP and reversal by sildenafil (a PDE5A inhibitor) corroborated the in vivo pathway involvement.
• This study extended the mechanistic understanding by showing circulating factors alone are sufficient to replicate this pathway dysregulation in isolated cardiomyocytes.
• Sildenafil 'completely reversed' the cGMP changes, consistent with PDE5A inhibition restoring cGMP signaling.