Use of Quadruple-Parametric Optical Mapping of a Mouse Heart to Measure Metabolism-Excitation-Contraction Coupling Changes with Pharmacological Interventions

Cardiovascular diseases are complex and alteration in many aspects of cardiac function can occur simultaneously. To study such complex diseases, we developed a quadruple mapping protocol which is an advancement on the previously published triple mapping publication. For decades, mechanical and electrical activity have been measured in the heart. However, more recently the combined measurement of metabolism with mechanical and electrical activity to measure the trifecta of cardiac function, known as metabolism-excitation-contraction-coupling (MECC) has been reported. In this study, we developed and validated the quadruple-parametric optical mapping technique and simultaneously image membrane potential, calcium handling, NADH, and FAD. We hypothesize that the calcium and sodium channel blocker used will alter their respective parameters with optical mapping. The four parameters of cardiac function were measured with drug treatment, 1µM Nifedipine, an L-type calcium channel blocker, and 1µM flecainide, a sodium channel blocker. Male and female murine hearts (n=6) were Langendorff perfused, and mechanical function was arrested with BDM. Hearts were stained with RH237 and RHOD-2AM, voltage and calcium dyes, respectively and dyes were excited by 520 nm excitation high power LED light. A second excitation light at 365 nm was used to induce autofluorescence of NADH and FAD. The heart was paced with a platinum pacing wire and restitution was performed from 200 ms basic cycle length (BCL) up to loss of 1:1 capture. Simultaneous optical recordings of membrane potential, intracellular calcium, NADH and FAD were obtained and analyzed using Rhythm3.0 program on Matlab. Nifedipine significantly increased calcium rise time to 17.52 ± 3.06 ms compared to baseline measurements of 11.87 ± 1.49 ms and flecainide significantly increased voltage rise time to 13.25 ± 4.35 ms compared to baseline of 5.91 ± 1.03 ms at 150 ms BCL (p<0.05). These results are expected due to the L-type calcium channel and sodium channel blocking capabilities of nifedipine and flecainide, respectively. Nifedipine also increased calcium decay 37.87 ± 5.89 ms compared to control 30.75 ± 4.52 ms (p<0.05). Interestingly, nifedipine decreased (became more negative) the voltage calcium delay (metric of excitation contraction coupling) at 150 and 200 ms BCL compared to control (p<0.05). Flecainide decreased both transverse 0.28 ± 0.02 and longitudinal 0.35 ± 0.05 conduction velocity compared to control (0.39 ± 0.02, 0.51 ± 0.05, respectively, p<0.05). Interestingly, flecainide increased NADH intensity toward the end of the restitution protocol (60, 70 ms, p<0.05). Neither drug effected action potential or calcium transient duration, calcium decay, FAD, or redox ratio (FAD/NADH). Taken together, we used quadruple parametric optical mapping to not just demonstrate known cardiac drug response to nifedipine and flecainide, but also demonstrate novel aspects of cardiac function that are affected by these drugs. These additional and novel parameter modulation by the drugs would not have been identified without such a multi-parametric approach to assessing cardiac physiology. Completion of this study with multiparametric assessment of drug and disease response will help to further validate our quadruple mapping approach and allow us to better understand the interplay between electrical, mechanical, and metabolic function of the heart. Lastly, this technique can be used to better understand the ramifications of pharmacological interventions on MECC in the heart. American Heart Association Career Development Award (George, #935807), Cardiology NIH Training Grant (Mills, T32 HL129964) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.