Acute inhibition of lipolysis after myocardial infarction using a murine adipocyte specific inhibitory DREADD model improves cardiac systolic function

The systemic increase in circulating catecholamines and the subsequent β-adrenergic stimulation during and after myocardial infarction (MI) is one of the strongest stimuli for peripheral white adipose tissue (WAT) lipolysis. Elevated levels of free fatty acids are detrimental to the ischemic heart, therefore inhibition of peripheral adipose tissue lipolysis is a promising therapeutic target. In our study, we used a murine adipocyte specific inhibitory DREADD model to enable a spatio-temporal-specific inhibition of lipolysis during and after experimental cardiac ischemia.

To activate cre-recombinase that removes a loxP-floxed STOP cassette to enable DREADD expression, 10-weeks old male DREADD mice were injected for 5 consecutive days 4-hydroxytamoxifen intraperitoneally (500 µg/mice) followed by 2,5 weeks wash-out phase. The successful expression of DREADDs was confirmed by histological analysis, western blot and qPCR. Experimental myocardial infarction was then induced according to the closed-chest-ischemia-protocol, where animals undergo first pre-surgery to induce a ligature around the LAD without ligation and 5-7 days afterwards 60 minutes ischemia by ligating the LAD without re-opening the chest. The activation of the adipocyte specific inhibitory DREADD by DREADD Agonist 21 before and after ischemia successfully reduced circulating NEFA levels after 30 min of reperfusion (DREADD -: BL: 0,195 ± 0,019 mmol/L, 30’ rep: 0,589 ± 0,069 mmol/L; DREADD +: BL: 0,195 ± 0,021 mmol/L, 30’ rep: 0,336 ± 0,033 mmol/L, n = 11-15, p = 0,0143). NEFAs are known to induces insulin resistance. In line with reduced NEFA levels multiplex analysis revealed a reduction also in circulating insulin levels after 30 min of reperfusion (DREADD-: 4762 ± 137,6 pg/mL; DREADD+: 2473 ± 67,3 pg/mL, n = 10-11, p < 0,001),

The acute inhibition of lipolysis in DREADD expressing mice lead to an improved systolic pump function after 7 days of reperfusion, as depicted by an increased ejection fraction (DREADD -: 31,9 ± 6,7 %; DREADD +: 40,2 ± 6,7 %, n = 11-13, p = 0,004), fractional area change (DREADD -: 22,7 ± 8,3 %; DREADD +: 34,1 ± 9,8 %, n = 11-13, p = 0,004) and stroke volume (DREADD -: 38,4 ± 9,6 µL; DREADD +: 47,2 ± 6,7 µL, n = 11-13, p = 0,0029). While scar size was unchanged after 7d of reperfusion (p=0,1849), strain analysis showed an improved strain and strain rate in the basal wall segments of the left ventricle, indicating an improved contractile function of the remote myocardium. Western Blot analysis using a broad phospho-PKA substrates antibody pointed to a higher PKA-activity in the heart of DREADD expressing animals after 30 min of reperfusion. A more detailed analysis of specific PKA targets revealed indeed a higher phosphorylation of phospholamban (DREADD-: 1 ± 0,1576; DREADD+: 1,814 ± 0,2886, n = 6-7, p = 0,0257) as well as troponin I (DREADD-: 1 ± 0,0613; DREADD+: 1,221 ± 0,0971, n = 7, p = 0,0786) in remote area.

Taken together these data show that the acute inhibition of peripheral lipolysis during and shortly after cardiac ischemia improves the remote myocardium function and link for the first time lipolytic activation and elevated NEFA level to cardiac PKA-activity and contractile function.