Regulation of ATP production by mitochondria, critical to multicellular life, is poorly understood.Here we investigate the molecular controls of this process in heart and provide a framework for its Ca 2+ -dependent regulation. We find that the entry of Ca 2+ into the matrix through the mitochondrial calcium uniporter (MCU) in heart has neither an apparent cytosolic Ca 2+ threshold nor gating function and guides ATP production by its influence on the inner mitochondrial membrane (IMM) potential, ΔΨ m . This regulation occurs by matrix Ca 2+ -dependent modulation of pyruvate and glutamate dehydrogenase activity and not through any effect of Ca 2+ on ATP Synthase or on Electron Transport Chain Complexes II, III or IV. Examining the ΔΨ m dependence of ATP production over the range of −60 mV to −170 mV in detail reveals that cardiac ATP synthase has a voltage dependence that distinguishes it fundamentally from the previous standard, the bacterial ATP synthase. Cardiac ATP synthase operates with a different ΔΨ m threshold for ATP production than bacterial ATP synthase and reveals a concave-upwards shape without saturation. Skeletal muscle MCU Ca 2+ flux, while also having no apparent cytosolic Ca 2+ threshold, is substantially different from the cardiac MCU, yet the ATP synthase voltage dependence in skeletal muscle is identical to that in the heart. These results suggest that while the conduction of cytosolic Ca 2+ signals through the MCU appears to be tissue-dependent, as shown by earlier work 1 , the control of ATP synthase by ΔΨ m appears to be broadly consistent among tissues but is clearly different from bacteria.

A gentle optical examination of the mitochondrial permeability transition pore (mPTP) opening events was carried out in isolated quiescent ventricular myocytes by tracking the inner membrane potential (ΔΨ M) using TMRM (tetramethylrhodamine methyl ester). Zeiss Airyscan 880 "superresolution" or "high-resolution" imaging was done with very low levels of illumination (0.009% laser power). In cellular areas imaged every 9 seconds (ROI or regions of interest), transient depolarizations of variable amplitudes occurred at increasing rates for the first 30 minutes. The time to first depolarization events was 8.4 min (± 1.1 SEM n=21 cells). At longer times, essentially permanent and irreversible depolarizations occurred at an increasing fraction of all events. In other cellular areas surrounding the ROI, mitochondria were rarely illuminated (once per 5 minutes) and virtually no permanent depolarization events occurred for over 1 hour of imaging. These findings suggest that photon stress due to the imaging itself plays an important role in the generation of both the transient mPTP opening events as well as the permanent mPTP opening events. Consistent with the evidence that photon "stress" in mitochondria loaded with virtually any photon absorbing substance, generates reactive oxygen species (ROS) [1-5], we show that cyclosporine-A (CsA, 10 mM) and the antioxidant n-acetyl cysteine (NAC, 10 mM), reduced the number of events by 80 % and 93% respectively. Furthermore, CsA and NAC

Calcium-induced calcium release is the principal mechanism that triggers the cell-wide [Ca2+]i transient that activates muscle contraction during cardiac excitation-contraction coupling (ECC). Here, we characterize this process in mouse cardiac myocytes with a novel mathematical action potential (AP) model that incorporates realistic stochastic gating of voltage-dependent L-type calcium (Ca2+) channels (LCCs) and sarcoplasmic reticulum (SR) Ca2+ release channels (the ryanodine receptors, RyR2s). Depolarization of the sarcolemma during an AP stochastically activates the LCCs elevating subspace [Ca2+] within each of the cell’s 20,000 independent calcium release units (CRUs) to trigger local RyR2 opening and initiate Ca2+ sparks, the fundamental unit of triggered Ca2+ release. Synchronization of Ca2+ sparks during systole depends on the nearly uniform cellular activation of LCCs and the likelihood of local LCC openings triggering local Ca2+ sparks (ECC fidelity). The detailed design and true SR Ca2+ pump/leak balance displayed by our model permits investigation of ECC fidelity and Ca2+ spark fi-delity, the balance between visible (Ca2+ spark) and invisible (Ca2+ quark/sub-spark) SR Ca2+ release events. Excess SR Ca2+ leak is examined as a disease mechanism in the context of “catecholaminergic polymorphic ventricular tachycardia (CPVT)”, a Ca2+-dependent arrhythmia. We find that that RyR2s (and therefore Ca2+ sparks) are relatively insensitive to LCC openings across a wide range of membrane potentials; and that key differences exist between Ca2+ sparks evoked during quiescence, diastole, and systole. The enhanced RyR2 [Ca2+]i sensitivity during CPVT leads to increased Ca2+ spark fidelity resulting in asynchronous systolic Ca2+ spark activity. It also produces increased diastolic SR Ca2+ leak with some prolonged Ca2+ sparks that at times become “metastable” and fail to efficiently terminate. There is a huge margin of safety for stable Ca2+ handling within the cell and this novel mechanistic model provides insight into the molecular signaling characteristics that help maintain overall Ca2+ stability even under the conditions of high SR Ca2+ leak during CPVT. Finally, this model should provide tools for investigators to examine normal and pathological Ca2+ signaling characteristics in the heart.

Mena, a member of the Ena/VASP family of actin regulatory proteins, modulates microfilaments and interacts with cytoskeletal proteins associated with heart failure. Mena is localized at the intercalated disc (ICD) of adult cardiac myocytes, colocalizing with numerous cytoskeletal proteins. Mena's role in the maintainence of mechanical myocardial stability at the cardiomyocyte ICD remains unknown. We hypothesized that Mena may modulate signals from the sarcolemma to the actin cytoskeleton at the ICD to regulate the expression and localization of connexin 43 (Cx43). The small GTPase Rac1 plays a pivotal role in the regulation of actin cytoskeletal reorganization and mediating morphological and transcriptional changes in cardiomyocytes. We found that Mena is associated with active Rac1 in cardiomyocytes and that RNAi knockdown of Mena increased Rac1 activity significantly. Furthermore, Mena knockdown increased Cx43 expression and altered Cx43 localization and trafficking at the ICD, concomitant with faster intercellular communication, as assessed by dye transfer between cardiomyocyte pairs. In mice overexpressing constitutively active Rac1, left ventricular Mena expression was increased significantly, concomitant with lateral redistribution of Cx43. These results suggest that Mena is a critical regulator of the ICD and is required for normal localization of Cx43 in part via regulation of Rac1.